Anti-TIGIT Antibodies

ABSTRACT

Anti-TIGIT antibodies and antigen binding fragments thereof that inhibit TIGIT-mediated signalling are provided, together with combinations comprising said antibodies or antigen binding fragments thereof and methods for their use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/872,494, filed Jul. 25, 2022, which is a continuation of U.S. patentapplication Ser. No. 16/413,557, filed May 15, 2019, now U.S. Pat. No.11,439,705, which is a continuation of U.S. patent application Ser. No.16/159,506, filed Oct. 12, 2018, now U.S. Pat. No. 10,329,349, which isa continuation of International Patent Application No.PCT/US2018/043968, filed Jul. 26, 2018, which claims the benefit of U.S.Provisional Application No. 62/660,640, filed on Apr. 20, 2018, U.S.Provisional Application No. 62/606,159, filed on Jul. 27, 2017, EPPatent Application No. 17184102.6 filed on Jul. 31, 2017, and BE PatentApplication No. 2017/5535 filed on Jul. 31, 2017, all of which areincorporated herein by reference in their entireties for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing with 370 sequenceswhich has been filed electronically in XML file format and is herebyincorporated by reference in its entirety. Said XML copy, created onAug. 31, 2022, is named 70097US06_SL.xml and is 340,057 bytes in size.

BACKGROUND

Cancer immunotherapy relies on the modulation of the immune system toincrease recognition and response against tumour cells. Such modulationcan be achieved by multiple mechanisms including the activation ofco-stimulatory molecules present on immune cells or through theinhibition of co-inhibitory receptors. The activation of an immuneresponse is a complex mechanism involving numerous cell populations likeantigen-presenting cells important for the initiation of theantigen-specific response and effector cells responsible for tumour celldestruction. The mechanisms modulating the activity of effector cellslike cytotoxic T cells are numerous and represent target of choice inthe context of cancer immunotherapy.

TIGIT (T cell Immunoreceptor with Ig and ITIM domains), also calledWUCAM, VSIG9 or Vstm3, is a co-inhibitory receptor preferentiallyexpressed on NK, CD8+ and CD4+ T cells as well as on regulatory T cells(Treg cells, or simply “Tregs”). TIGIT is transmembrane proteincontaining a known ITIM domain in its intracellular portion, atransmembrane domain and an immunoglobulin variable domain on theextracellular part of the receptor. Several ligands were described tobind to TIGIT receptor with CD155/PVR showing the best affinity followedby CD113/PVRL3 and CD112/PVRL2 (Yu et al. (2009) Nat. Immunol. 10:48.).DNAM/CD226, a known co-stimulatory receptor also expressed on NK and Tcells competes with TIGIT for CD155 and CD112 binding but with a loweraffinity, which suggests a tight control of the activation of theseeffector cells to avoid uncontrolled cytotoxicity against normal cellsexpressing CD155 ligand.

TIGIT expression is increased on tumour infiltrating lymphocytes (TILs)and in disease settings such as HIV infection. TIGIT expression marksexhausted T cells that have lower effector function as compared to TIGITnegative counterparts (Kurtulus et al. (2015) J. Clin. Invest. 276:112;Chew et al. (2016) Plos Pathogens. 12). Conversely, Treg cells thatexpress TIGIT show enhanced immunosuppressive activity as compared toTIGIT negative Treg population (Joller et al. (2014) Immunity. 40:569).

Like other co-inhibitory receptors (PD1 or CTLA4) expressed on T cellsthat have been proven to be relevant target for immunotherapy and forwhich antagonistic antibodies have been approved for the treatment ofhuman cancer, the development of antagonistic anti-TIGIT antibody mayhelp to turn-on the immune system and better fight cancer cells. It hasbeen suggested that antagonistic anti-TIGIT antibodies in monotherapy orin combination with a-PD1 antibody could achieve strong anti-tumourefficacy in preclinical models (Johnston et al. (2014) Cancer Cell 26:1;WO2016/028656; US2016/0176963; US2016/0376365, all of which areincorporated herein by reference).

Thus, antagonistic antibodies specific for TIGIT that could inhibitTIGIT receptor activity represent an opportunity to decrease theimmunosuppressive effect associated with tumour microenvironments andthereby increase antitumor immune response against tumour cells.

SUMMARY OF INVENTION

The present invention provides anti-TIGIT antibodies that can decreasethe immunosuppressive effect of TIGIT-mediated signalling. Inparticular, antibodies or antigen binding fragments of the invention caninhibit TIGIT-mediated immunosuppression through prevention of ligandbinding on T cells (conventional αβ T cells and non-conventional γδ Tcells) and NK cells and/or depletion of TIGIT positive Treg cells,and/or by inducing internalisation of the TIGIT receptor.

In one aspect, the present invention provides an isolated antibody orantigen binding fragment thereof which binds to human TIGIT and whichcomprises a heavy chain variable domain comprising a heavy chain CDR1(HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3)selected from the HCDR1, HCDR2 and HCDR3 sequences shown in FIG. 1 andwhich further comprises a light chain variable domain comprising a lightchain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3(LCDR3) selected from the LCDR1, LCDR2, and LCDR3 sequences shown inFIG. 2 .

In certain embodiments the antibody or antigen binding fragmentcomprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3,wherein the combination is selected from the group of combinationsformed by the HCDRs from each antibody in FIG. 1 taken with the LCDRsfrom the corresponding antibody in FIG. 2 .

In certain embodiments, an antibody or antigen binding fragmentaccording the invention may comprise a heavy chain variable domainhaving an amino acid sequence selected from the group consisting of: SEQID Nos: 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 239, 327, 329, and 331 and amino acid sequences exhibiting at least90%, 95%, 97%, 98% or 99% sequence identity thereto; and optionallycomprise a light chain variable domain having an amino acid sequenceselected from the group consisting of: the amino acid sequences of SEQID Nos: 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 328, 330, and 332 and amino acid sequences exhibiting at least90%, 95%, 97%, 98% or 99% sequence identity thereto.

In certain embodiments the antibody or antigen binding fragmentcomprises a combination of a heavy chain variable domain and a lightchain variable domain, wherein the combination is selected from thegroup of combinations formed by the VH from each antibody in FIG. 5 , oran amino acid sequence exhibiting at least 90%, 95%, 97%, 98% or 99%sequence identity thereto, taken with the VL from the same antibody inFIG. 5 , or an amino acid sequence exhibiting at least 90%, 95%, 97%,98% or 99% sequence identity thereto.

The most-preferred antibodies and antigen binding fragments providedherein are those based on the CDRs or complete variable domains ofantibody 31282 provided herein.

As demonstrated herein, these preferred anti-TIGIT antibodies andantigen binding fragments based on antibody 31282 have particularlysurprising and advantageous properties. These properties include: ahigher affinity for TIGIT expressed on CD8 T cells (from healthy donorsor from cancer patients) compared to each previously describedanti-TIGIT antibody tested; a better IC50 for competition with CD155/PVRcompared to each previously described anti-TIGIT antibody tested; abetter EC50 in T cell activation assays compared to each previouslydescribed anti-TIGIT antibody tested; and potently increasing activityin T cells from cancer patient peripheral blood, and importantly intumour infiltrating lymphocytes. Furthermore, it is surprisingly shownherein that antibodies and antigen binding fragments according to theinvention, especially those based on antibody 31282, preferentiallydeplete Treg cells. That is, TIGIT-expressing Treg cells exposed to theprovided anti-TIGIT antibodies undergo lysis to a greater proportioncompared to conventional CD4 and CD8 T cells. This is surprising becauseconventional CD4 and CD8 T cells also express TIGIT, but do not undergocell lysis to the same extent when contacted with the antibodies. It isfurther surprisingly shown that antibodies and antigen binding fragmentsaccording to the invention, especially those based on antibody 31282 notonly promote conventional T cell pro-inflammatory activity, but alsoincrease activity of non-conventional γδ T cells.

Thus, in certain preferred embodiments, provided herein is an antibodyor antigen binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 wherein:

HCDR1 comprises or consists of SEQ ID NO: 16 (YTFTSYYMH),HCDR2 comprises or consists of SEQ ID NO: 17 (VIGPSGASTSYAQKFQG),HCDR3 comprises or consists of SEQ ID NO: 18 (ARDHSDYWSGIMEV),LCDR1 comprises or consists of SEQ ID NO: 61 (RASQSVRSSYLA),LCDR2 comprises or consists of SEQ ID NO: 62 (GASSRAT), andLCDR3 comprises or consists of SEQ ID NO: 63 (QQYFSPPWT).

In certain such embodiments, the heavy chain variable domain comprisesor consists of an amino acid sequence according to SEQ ID NO: 221 or anamino acid sequence exhibiting at least 90%, 95%, 97%, 98% or 99%sequence identity thereto, and the light chain variable domain comprisesor consists of an amino acid sequence according to SEQ ID NO: 222 or anamino acid sequence exhibiting at least 90%, 95%, 97%, 98% or 99%sequence identity thereto.

In certain preferred embodiments the anti-TIGIT antibody is antibody31282 described herein.

In a further aspect the invention provides an isolated antibody orantigen binding fragment thereof, which cross-competes for binding tohuman TIGIT with an antibody according to the first aspect of theinvention, for example an antibody exemplified herein.

In a further aspect, the invention provides an isolated antibody orantigen binding fragment thereof, which binds to the same epitope as anantibody according to the first aspect of the invention, for example anantibody exemplified herein.

In a further aspect, the invention provides an antibody or antigenbinding fragment thereof which binds to an epitope of human TIGITcomprising TIGIT residues Q56, and I109, optionally comprising residuesQ56, N58 and I109. In preferred embodiments is provided an antibody orantigen binding fragment thereof which binds to an epitope of humanTIGIT comprising TIGIT residues Q56, N58, E60, I68, L73, H76, and I109.

In certain embodiments, the antibody or antigen binding fragment thereofbinds to an epitope of human TIGIT consisting of TIGIT residues Q56,N58, E60, I68, L73, H76, and I109.

In a further aspect, the invention provides an isolated antibody orantigen binding fragment thereof which binds to human TIGIT and whichdoes not compete with CD155 for TIGIT binding.

In certain embodiments, the antibody or antigen binding fragment whichbinds to human TIGIT and which does not compete with CD155 for TIGITbinding comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 whereinHCDR1 comprises or consists of SEQ ID NO: 280, HCDR2 comprises orconsists of SEQ ID NO: 281, HCDR3 comprises or consists of SEQ ID NO:282, and LCDR1 comprises or consists of SEQ ID NO: 292, LCDR2 comprisesor consists of SEQ ID NO: 293, and LCDR3 comprises or consists of SEQ IDNO: 294.

In certain such embodiments, the heavy chain variable domain comprisesor consists of the amino acid sequence shown as SEQ ID NO: 333 or anamino acid sequence exhibiting at least 90%, 95%, 97%, 98% or 99%sequence identity thereto, and the light chain variable domain comprisesor consists of the amino acid sequence shown as SEQ ID NO: 334 or anamino acid sequence exhibiting at least 90%, 95%, 97%, 98% or 99%sequence identity thereto.

In certain preferred embodiments, the antibody which binds to humanTIGIT and which does not compete with CD155 for TIGIT binding comprisesa heavy chain variable domain and a light chain variable domain whereinHCDR1 comprises SEQ ID NO: 353, HCDR2 comprises SEQ ID NO: 354, HCDR3comprises SEQ ID NO: 355, and LCDR1 comprises SEQ ID NO: 356, LCDR2comprises SEQ ID NO: 357, and LCDR3 comprises SEQ ID NO: 358.

In certain such embodiments, the heavy chain variable domain maycomprise the amino acid sequence shown as SEQ ID NO: 367 or an aminoacid sequence exhibiting at least 90%, 95%, 97%, 98% or 99% sequenceidentity thereto, and the light chain variable domain may comprise theamino acid sequence shown as SEQ ID NO: 368 or an amino acid sequenceexhibiting at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

In a further aspect, the invention provides an isolated anti-TIGITantibody or antigen binding fragment thereof which preferentiallydepletes TIGIT-expressing Treg cells, optionally wherein the antibody orantigen binding fragment is an antibody or antigen binding fragmentaccording to the first aspect of the invention, for example an antibodyexemplified herein.

In a further aspect the invention provides an affinity variant of anantibody according to other aspects of the invention, for example anantibody exemplified herein.

In a further aspect the invention provides an isolated polynucleotide orcombination of isolated polynucleotides encoding an antibody or antigenbinding fragment according to any other aspect of the invention, forexample an antibody exemplified herein.

In a further aspect the invention provides an isolated polynucleotideencoding a VH and/or a VL domain of an anti-TIGIT antibody, wherein thepolynucleotide comprises one or more sequences selected from the groupconsisting of SEQ ID Nos: 241-270, 335-342 and 369-370.

In a further aspect the invention provides an expression vectorcomprising a polynucleotide or combination of polynucleotides accordingto the invention operably linked to regulatory sequences which permitexpression of the antigen binding polypeptide in a host cell orcell-free expression system.

In a further aspect the invention provides a host cell or cell-freeexpression system containing an expression vector according to theinvention.

In a further aspect the invention provides a method of producing arecombinant antibody or antigen binding fragment thereof which comprisesculturing a host cell or cell free expression system according to theinvention under conditions which permit expression of the antibody orantigen binding fragment and recovering the expressed antibody orantigen binding fragment.

In a further aspect the invention provides a pharmaceutical compositioncomprising an antibody or antigen binding fragment according to theinvention, for example an antibody exemplified herein, and at least onepharmaceutically acceptable carrier or excipient.

In a further aspect the invention provides an antibody orantigen-binding fragment according to the invention or pharmaceuticalcomposition according to the invention for use in therapy.

In a further aspect, the invention provides an antibody orantigen-binding fragment according to the invention (for example anantibody exemplified herein) or pharmaceutical composition according tothe invention for use in a method of treating cancer.

In a further aspect, the invention provides an antibody orantigen-binding fragment according to the invention (for example anantibody exemplified herein) or pharmaceutical composition according tothe invention for use in a method of treating viral infection,optionally CMV infection.

In a further aspect the invention provides a method of treating cancerin a subject comprising administering an effective amount of an antibodyor antigen-binding fragment according to the invention (for example anantibody exemplified herein) or pharmaceutical composition according tothe invention to the subject, thereby treating the cancer.

In a further aspect is provided a method of treating viral infection ina subject comprising administering an effective amount of an antibody orantigen-binding fragment according to the invention or pharmaceuticalcomposition according to the invention to the subject, thereby treatingthe viral infection. In preferred embodiments the viral infection is CMVinfection.

In a further aspect is provided a method of promoting T cell activitycomprising contacting a population of T cells with an antibody orantigen binding fragment according to the invention. In certainembodiments the method promotes αβ T cell activity. In certainembodiments the method promotes γδ T cell activity. In certainembodiments the method is performed in vitro. In certain embodiments themethod is performed in vivo, for example in a human subject.

In certain embodiments is provided a method according to the invention,or an antibody or antigen-binding fragment or pharmaceutical compositionfor use in a method according to the invention, wherein the methodfurther comprises administration of one or more additional therapeuticagents. In certain preferred embodiments, the one or more additionalagents are selected from: a chemotherapeutic agent, an anti-PD1antibody, an anti-PD-L1 antibody, an anti-41BB antibody, an anti-OX40antibody, an anti-GITR antibody, and an anti-ICOS antibody.

In a further aspect is provided a combination comprising an anti-TIGITantibody or antigen binding fragment thereof and one or more of achemotherapeutic agent, an anti-PD1 antibody, an anti-PD-L1 antibody, ananti-41BB antibody, an anti-OX40 antibody, an anti-GITR antibody, and ananti-ICOS antibody. In a further aspect is provided a combinationaccording to the invention for use in therapy. In a further aspect isprovided a combination according to the invention for use in a method oftreating cancer or for use in a method of treating viral infection. In afurther aspect is provided a combination according to the invention foruse in a method according to the invention. In a preferred embodimentthe anti-TIGIT antibody or antigen binding fragment thereof is anantibody of the invention or an antigen binding fragment thereof.

In all relevant aspects, it is preferred that any subject to be treatedis a human subject. In all relevant aspects it is preferred that cells(e.g. T cells) contacted with antibodies according to the invention arehuman cells (e.g. human T cells).

Unless technically incompatible or indicated to the contrary, anypreferred embodiment described can optionally be used in combinationwith one or more of all other preferred embodiments.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 : Table providing heavy chain variable domain (VH)complementarity determining region (CDR) sequences of antibodies of theinvention.

FIG. 2 : Table providing light chain variable domain (VL) CDR sequencesof antibodies of the invention.

FIG. 3 : Table providing heavy chain variable domain (VH) framework (FR)sequences of antibodies of the invention.

FIG. 4 : Table providing light chain variable domain (VL) framework (FR)sequences of antibodies of the invention.

FIG. 5 : Table providing heavy chain variable domain (VH) and lightchain variable domain (VL) amino acid sequences of antibodies of theinvention.

FIG. 6 : Table providing sequences of polynucleotides encoding VH and VLdomains of antibodies according to the invention.

FIG. 7 : Graph showing the results of a competition assay between hCD155and anti-TIGIT antibody for binding to Jurkat-hTIGIT.

FIG. 8A: Graph showing the proportion of TIGIT positive cells withinspecific T cell populations of PBMC from 7 healthy human donors. FIG.8B: Graph showing the proportion of TIGIT positive cells withindifferent immune populations of PBMC from 7 healthy human donors.

FIG. 9 : Graph showing the results of a binding assay of anti-TIGITantibody on Jurkat-hTIGIT.

FIG. 10A and FIG. 10B: Graphs showing the results of a binding assay ofanti-TIGIT antibody on primary CD8+ T cells from human healthy PBMCs.FIG. 10C Graph showing the results of a binding assay of anti-TIGITantibody on primary memory CD8+ T cells and Treg from human healthyPBMCs.

FIG. 11A and FIG. 11B: Graphs showing the results of a binding assay ofanti-TIGIT antibody on primary CD8+ T cells from cynomolgus healthyPBMCs.

FIG. 12A, FIG. 12B, and FIG. 12C: Graphs showing the effect ofanti-TIGIT antibodies in a CHO-TCR-CD155 and Jurkat-hTIGIT Bioassay.

FIG. 13A, FIG. 13B, FIG. 13C: Graphs showing the effect of anti-TIGITantibodies to increase IFNg secretion in a functional assay on humanprimary CD8 T cells from healthy donors activated with CHO-TCR-CD155cells.

FIG. 14 : Histogram plots showing the effect of anti-TIGIT antibody toincrease IFNg secretion in a functional assay on human primary CD8+ TILsfrom an ovarian ascites activated with CHO-TCR-CD155 cells.

FIG. 15A Graph showing the results of a competition assay between mouseCD155 and anti-TIGIT antibody for binding to Jurkat-mTIGIT. FIG. 15BGraph showing the effect of anti-TIGIT antibody to increase IFNgsecretion in a functional assay on mouse OT-1 T cells. FIG. 15C Graphshowing the effect of anti-TIGIT antibody to increase cytotoxicity in afunctional assay on mouse OT-1 T cells.

FIG. 16A Graph showing the anti-tumor efficacy of anti-TIGIT antibody inmonotherapy in a CT26 tumor model. FIG. 16B and FIG. 16C Graphs showingthe anti-tumor efficacy of anti-TIGIT antibody in combination withanti-PD1 in a CT26 tumor model.

FIG. 17A Graph showing the isotype dependent anti-tumor efficacy ofanti-TIGIT antibody in monotherapy in a CT26 tumor model. FIG. 17B Graphshowing the isotype dependent anti-tumor efficacy of anti-TIGIT antibodyin combination with anti-PD1 in a CT26 tumor model.

FIG. 18A and FIG. 18G Graphs showing the modulation of proportion ofTreg cell within total CD4+ T cell population in CT26 tumor treated withanti-TIGIT antibody in monotherapy or combination with anti-PD1. FIG.18B and FIG. 18H Graphs showing the modulation of proportion of CD8+ Tcell within total CD45+ population in CT26 tumor treated with anti-TIGITantibody in monotherapy or combination with anti-PD1. FIG. 18C and FIG.18I Graphs showing the modulation of CD8+/Treg T cell ratio in CT26tumor treated with anti-TIGIT antibody in monotherapy or combinationwith anti-PD1. FIG. 18D and FIG. 18J Graph showing the modulation ofIFNg secreting CD4+ T cells in CT26 tumor treated with anti-TIGITantibody in monotherapy or combination with anti-PD1. FIG. 18E Graphshowing the modulation of IFNg secreting CD8+ T cells in CT26 tumortreated with anti-TIGIT antibody. FIG. 18L and FIG. 18F Graphs showingthe ratio of IFNg/IL-10 secreting CD4+ T cells in CT26 tumor treatedwith anti-TIGIT antibody in monotherapy or combination with anti-PD1.FIG. 18K Graph showing the modulation of IL-10 secreting CD4+ T cells inCT26 tumor treated by anti-TIGIT antibody in combination with anti-PD1antibody.

FIG. 19A Volcano plot showing the effect of anti-TIGIT antibodytreatment to modulate gene expression in CT26 tumor and measured byNanoString analysis. FIG. 19B Box plot showing the modulation ofcytotoxic score in CT26 tumor treated with anti-TIGIT antibody inmonotherapy or combination with anti-PD1. FIG. 19C Box plot showing themodulation of CD8+ T cell score in CT26 tumor treated with anti-TIGITantibody in monotherapy or combination with anti-PD1.

FIG. 20A Histogram plots showing the proportion of TIGIT+ CD4+, CD8+ Tcell and Treg populations in PBMC from human healthy volunteers. FIG.20B Graph showing the in vitro cytotoxicity effect of anti-TIGITantibody on conventional CD4+, CD8+ T cell and Treg populations in PBMCfrom human healthy volunteers.

FIG. 21 Graph showing the ex-vivo cytotoxicity effect of anti-TIGITantibody on conventional CD4+, CD8+ T cell and Treg populations in CT26tumour.

FIG. 22A Graph showing the results of a binding assay of anti-TIGITantibody clones on Jurkat-hTIGIT cells. FIG. 22B Graph showing theresults of a binding assay of anti-TIGIT antibody clones on primary CD8+T cells from healthy human PBMCs. FIG. 22C Graph showing the results ofa binding assay of anti-TIGIT antibody clones on primary CD8+ T cellsfrom cancer patients PBMCs.

FIG. 23 Graph showing the results of a competition assay between humanCD155 and anti-TIGIT antibody clones for binding to Jurkat-hTIGIT.

FIGS. 24A-C Graphs showing the functional characterization of antagonista-TIGIT clones. FIG. 24A Graph showing the effect of anti-TIGITantibodies in a functional assay using Jurkat-hTIGIT effector cells(Luciferase reporter assay). FIG. 24B Graph showing the effect ofanti-TIGIT antibodies in a functional assay measuring IFNg secretion byhuman primary CD8+ T cell from healthy volunteers. FIG. 24C Graphshowing the effect of anti-TIGIT antibody clone 31282 in functionalassay measuring IFNg secretion by cancer patient CD3+ T cell from PBMC.FIG. 24D Graph showing the effect of anti-TIGIT antibody clone 31282 infunctional assay measuring intracellular cytokine staining in cancerpatient TILs or PBMCs.

FIG. 25 Cytotoxic activity of a-TIGIT clone 31282 on total memory CD4+or CD8+ T cells and Treg populations in PBMC from cancer patient.

FIGS. 26A-26B Graphs showing the characterization of TIGIT expression onimmune populations from cancer patients. FIG. 26A Frequency of TIGITexpression on immune populations from cancer patient PBMC and TILs. FIG.26B Absolute quantification of TIGIT expression on immune populationsfrom cancer patient PBMC and TILs.

FIG. 27A Structure of the Fab:TIGIT complex shown as ribbon diagram;FIG. 27B Full binding interface between clone 31282 and TIGIT; FIG. 27CBinding interface between clone 31282 and TIGIT showing contactedresidues.

FIG. 28 Competition assay between anti-TIGIT clones 31282 and 32959.

FIG. 29 Measure of plasma concentration of anti-TIGIT clone 31282 afteri.v. injection of a single dose at 0.1 mg/kg (top row), 1 mg/kg (middlerow) or 10 mg/kg (bottom row) in Cynomolgus monkey. Left column: 31282IgG1; right column 31282 IgG4.

FIGS. 30A-30B Plots showing the characterization of TIGIT expression onmalignant and normal CD4+ T cell populations from patient with SézarySyndrome. FIG. 30A Gating strategy to separate malignant and normal CD4+T cells. FIG. 30B MFI for TIGIT staining on the 2 distinct populations.

FIGS. 31A-31B Plots showing the characterization of TIGIT expression onmalignant and normal B cell populations from patient with CLL. FIG. 31AGating strategy to separate malignant and normal B cells. FIG. 31B MFIfor TIGIT staining on the 2 distinct populations.

FIGS. 32A-32C Graph showing the tumor growth curves in mice inoculatedwith EL4-mTIGIT tumors. FIG. 32A Median tumor growth curves. FIG. 32BIndividual tumor growth curves in mice treated with hIgG1 isotypecontrol antibody. FIG. 32C Individual tumor growth curves in micetreated with mouse surrogate antagonist a-TIGIT antibody (hIgG1). FIGS.32D-32F Graph showing the tumor growth curves in mice inoculated withEL4-GFP tumors. FIG. 32D Median tumor growth curves. FIG. 32E Individualtumor growth curves in mice treated with hIgG1 isotype control antibody.FIG. 32F Individual tumor growth curves in mice treated with surrogateantagonist a-TIGIT (hIgG1).

FIGS. 33A-33D Graphs showing the tumor growth curves in mice inoculatedwith CT26 tumors. FIG. 33A Median and individual tumor growth curves formice treated with anti-TIGIT and anti-4-1BB antibodies. FIG. 33B Medianand individual tumor growth curves for mice treated with anti-TIGIT andanti-OX-40 antibodies. FIG. 33C Median and individual tumor growthcurves for mice treated with anti-TIGIT and anti-GITR antibodies. FIG.33D Median and individual tumor growth curves for mice treated withanti-TIGIT and anti-ICOS antibodies.

FIG. 34A-34C Graphs showing the effect of anti-TIGIT antibodies on γδ Tcells. FIG. 34A Median proportion of TIGIT positive cells and TIGIT MFIsignal within Vδ2− γδ T cell populations of PBMC from CMV positive andnegative human donors. FIG. 34B Graph showing the activity of anti-TIGITAb to increase IFNg secretion in a functional assay on isolated humanprimary Vδ1+ γδ T cells. FIG. 34C Graph showing the activity ofanti-TIGIT Ab to increase IFNg secretion in a functional assay on totalPBMC.

DETAILED DESCRIPTION OF INVENTION

As used herein, the term “immunoglobulin” includes a polypeptide havinga combination of two heavy and two light chains whether or not itpossesses any relevant specific immunoreactivity. “Antibodies” refers tosuch assemblies which have significant known specific immunoreactiveactivity to an antigen of interest (e.g. TIGIT). The term “TIGITantibodies” or “anti-TIGIT antibodies” are used herein to refer toantibodies which exhibit immunological specificity for TIGIT protein.Antibodies and immunoglobulins comprise light and heavy chains, with orwithout an interchain covalent linkage between them. Basicimmunoglobulin structures in vertebrate systems are relatively wellunderstood.

The generic term “immunoglobulin” comprises five distinct classes ofantibody that can be distinguished biochemically. Although all fiveclasses of antibodies are within the scope of the present invention, thefollowing discussion will generally be directed to the IgG class ofimmunoglobulin molecules. With regard to IgG, immunoglobulins comprisetwo identical light polypeptide chains of molecular weight approximately23,000 Daltons, and two identical heavy chains of molecular weight53,000-70,000. The four chains are joined by disulfide bonds in a “Y”configuration wherein the light chains bracket the heavy chains startingat the mouth of the “Y” and continuing through the variable region.

The light chains of an antibody are classified as either kappa or lambda(κ, λ). Each heavy chain class may be bound with either a kappa orlambda light chain. In general, the light and heavy chains arecovalently bonded to each other, and the “tail” portions of the twoheavy chains are bonded to each other by covalent disulfide linkages ornon-covalent linkages when the immunoglobulins are generated by B cellsor genetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain. Thoseskilled in the art will appreciate that heavy chains are classified asgamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with somesubclasses among them (e.g., γ1-γ4). It is the nature of this chain thatdetermines the “class” of the antibody as IgG, IgM, IgA, IgD or IgE,respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2,IgG3, IgG4, IgA1, etc. are well characterized and are known to conferfunctional specialization. Modified versions of each of these classesand isotypes are readily discernible to the skilled artisan in view ofthe instant disclosure and, accordingly, are within the scope of theinstant invention.

As indicated above, the variable region of an antibody allows theantibody to selectively recognize and specifically bind epitopes onantigens. That is, the VL domain and VH domain of an antibody combine toform the variable region that defines a three dimensional antigenbinding site. This quaternary antibody structure forms the antigenbinding site present at the end of each arm of the Y. More specifically,the antigen binding site is defined by three complementary determiningregions (CDRs) on each of the VH and VL chains.

As used herein, the terms “TIGIT protein” or “TIGIT antigen” or “TIGIT”are used interchangeably and refer to the human T-cell immunoreceptor(GenBank accession number: NM 173799) that binds the poliovirus receptor(PVR—also known as CD155). TIGIT is also known as VSIG9, VSTM3, orWUCAM. Reference to TIGIT includes the native human TIGIT proteinnaturally expressed in the human host and/or on the surface of humancultured cell lines, as well as recombinant forms and fragments thereofand also naturally occurring mutant forms.

As used herein, the term “binding site” comprises a region of apolypeptide which is responsible for selectively binding to a targetantigen of interest (e.g. TIGIT). Binding domains comprise at least onebinding site. Exemplary binding domains include an antibody variabledomain. The antibody molecules of the invention may comprise a singlebinding site or multiple (e.g., two, three or four) binding sites.

As used herein the term “derived from” a designated protein (e.g. aTIGIT antibody or antigen-binding fragment thereof) refers to the originof the polypeptide. In one embodiment, the polypeptide or amino acidsequence which is derived from a particular starting polypeptide is aCDR sequence or sequence related thereto. In one embodiment, the aminoacid sequence which is derived from a particular starting polypeptide isnot contiguous. For example, in one embodiment, one, two, three, four,five, or six CDRs are derived from a starting antibody. In oneembodiment, the polypeptide or amino acid sequence which is derived froma particular starting polypeptide or amino acid sequence has an aminoacid sequence that is essentially identical to that of the startingsequence, or a portion thereof wherein the portion consists of at least3-5 amino acids, at least 5-10 amino acids, at least 10-20 amino acids,at least 20-30 amino acids, or at least 30-50 amino acids, or which isotherwise identifiable to one of ordinary skill in the art as having itsorigin in the starting sequence. In one embodiment, the one or more CDRsequences derived from the starting antibody are altered to producevariant CDR sequences, e.g. affinity variants, wherein the variant CDRsequences maintain TIGIT binding activity.

As used herein, a “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art, including basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in an immunoglobulin polypeptide may be replaced with anotheramino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from the constant domains of an immunoglobulin heavychain. A polypeptide comprising a heavy chain portion comprises at leastone of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hingeregion) domain, a CH2 domain, a CH3 domain, or a variant or fragmentthereof. In one embodiment, an antibody or antigen binding fragment ofthe invention may comprise the Fc portion of an immunoglobulin heavychain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). Inanother embodiment, an antibody or antigen binding fragment of theinvention may lack at least a portion of a constant domain (e.g., all orpart of a CH2 domain). In certain embodiments, at least one, andpreferably all, of the constant domains are derived from a humanimmunoglobulin heavy chain. For example, in one preferred embodiment,the heavy chain portion comprises a fully human hinge domain. In otherpreferred embodiments, the heavy chain portion comprises a fully humanFc portion (e.g., hinge, CH2 and CH3 domain sequences from a humanimmunoglobulin).

In certain embodiments, the constituent constant domains of the heavychain portion are from different immunoglobulin molecules. For example,a heavy chain portion of a polypeptide may comprise a CH2 domain derivedfrom an IgG1 molecule and a hinge region derived from an IgG3 or IgG4molecule. In other embodiments, the constant domains are chimericdomains comprising portions of different immunoglobulin molecules. Forexample, a hinge may comprise a first portion from an IgG1 molecule anda second portion from an IgG3 or IgG4 molecule. As set forth above, itwill be understood by one of ordinary skill in the art that the constantdomains of the heavy chain portion may be modified such that they varyin amino acid sequence from the naturally occurring (wild-type)immunoglobulin molecule. That is, the polypeptides of the inventiondisclosed herein may comprise alterations or modifications to one ormore of the heavy chain constant domains (CH1, hinge, CH2 or CH3) and/orto the light chain constant region domain (CL). Exemplary modificationsinclude additions, deletions or substitutions of one or more amino acidsin one or more domains.

As used herein, the terms “variable region” and “variable domain” areused interchangeably and are intended to have equivalent meaning. Theterm “variable” refers to the fact that certain portions of the variabledomains VH and VL differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody forits target antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called “hypervariable loops” in each of the VL domain andthe VH domain which form part of the antigen binding site. The first,second and third hypervariable loops of the VLambda light chain domainare referred to herein as L1(λ), L2(λ) and L3(λ) and may be defined ascomprising residues 24-33 (L1(λ), consisting of 9, 10 or 11 amino acidresidues), 49-53 (L2(λ), consisting of 3 residues) and 90-96 (L3(λ),consisting of 5 residues) in the VL domain (Morea et al., Methods 20,267-279, 2000). The first, second and third hypervariable loops of theVKappa light chain domain are referred to herein as L1(κ), L2(κ) andL3(κ) and may be defined as comprising residues 25-33 (L1(κ), consistingof 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(κ), consisting of 3residues) and 90-97 (L3(κ), consisting of 6 residues) in the VL domain(Morea et al., Methods 20, 267-279, 2000). The first, second and thirdhypervariable loops of the VH domain are referred to herein as H1, H2and H3 and may be defined as comprising residues 25-33 (H1, consistingof 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and91-105 (H3, highly variable in length) in the VH domain (Morea et al.,Methods 20, 267-279, 2000).

Unless otherwise indicated, the terms L1, L2 and L3 respectively referto the first, second and third hypervariable loops of a VL domain, andencompass hypervariable loops obtained from both VKappa and VLambdaisotypes. The terms H1, H2 and H3 respectively refer to the first,second and third hypervariable loops of the VH domain, and encompasshypervariable loops obtained from any of the known heavy chain isotypes,including γ, ε, δ, α or μ.

The hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise partof a “complementarity determining region” or “CDR”, as defined below.The terms “hypervariable loop” and “complementarity determining region”are not strictly synonymous, since the hypervariable loops (HVs) aredefined on the basis of structure, whereas complementarity determiningregions (CDRs) are defined based on sequence variability (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991) and thelimits of the HVs and the CDRs may be different in some VH and VLdomains.

The CDRs of the VL and VH domains can typically be defined as comprisingthe following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2) and89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or31-35b (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in the heavy chainvariable domain; (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991). Thus, the HVs may be comprised within thecorresponding CDRs and references herein to the “hypervariable loops” ofVH and VL domains should be interpreted as also encompassing thecorresponding CDRs, and vice versa, unless otherwise indicated.

The more highly conserved portions of variable domains are called theframework region (FR), as defined below. The variable domains of nativeheavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4,respectively), largely adopting a β-sheet configuration, connected bythe three hypervariable loops. The hypervariable loops in each chain areheld together in close proximity by the FRs and, with the hypervariableloops from the other chain, contribute to the formation of theantigen-binding site of antibodies. Structural analysis of antibodiesrevealed the relationship between the sequence and the shape of thebinding site formed by the complementarity determining regions (Chothiaet al., J. Mol. Biol. 227, 799-817, 1992; Tramontano et al., J. Mol.Biol, 215, 175-182, 1990). Despite their high sequence variability, fiveof the six loops adopt just a small repertoire of main-chainconformations, called “canonical structures”. These conformations arefirst of all determined by the length of the loops and secondly by thepresence of key residues at certain positions in the loops and in theframework regions that determine the conformation through their packing,hydrogen bonding or the ability to assume unusual main-chainconformations.

As used herein, the term “CDR” or “complementarity determining region”means the non-contiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616, 1977, by Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991, by Chothia et al., J. Mol.Biol. 196, 901-917, 1987, and by MacCallum et al., J. Mol. Biol. 262,732-745, 1996, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. The amino acidresidues which encompass the CDRs as defined by each of the above citedreferences are set forth for comparison. Preferably, the term “CDR” is aCDR as defined by Kabat based on sequence comparisons.

TABLE 1 CDR definitions. CDR Definitions Kabat¹ Chothia² MacCallum³V_(H) CDR1 31-35 26-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3 95-102  96-101  93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-5650-52 46-55 V_(L) CDR3 89-97 91-96 89-96 ¹Residue numbering follows thenomenclature of Kabat et al., supra ²Residue numbering follows thenomenclature of Chothia et al., supra ³Residue numbering follows thenomenclature of MacCallum et al., supra

As used herein, the term “framework region” or “FR region” includes theamino acid residues that are part of the variable region, but are notpart of the CDRs (e.g., using the Kabat definition of CDRs). Therefore,a variable region framework is between about 100-120 amino acids inlength but includes only those amino acids outside of the CDRs. For thespecific example of a heavy chain variable domain and for the CDRs asdefined by Kabat et al., framework region 1 corresponds to the domain ofthe variable region encompassing amino acids 1-30; framework region 2corresponds to the domain of the variable region encompassing aminoacids 36-49; framework region 3 corresponds to the domain of thevariable region encompassing amino acids 66-94, and framework region 4corresponds to the domain of the variable region from amino acids 103 tothe end of the variable region. The framework regions for the lightchain are similarly separated by each of the light claim variable regionCDRs. Similarly, using the definition of CDRs by Chothia et al. orMcCallum et al. the framework region boundaries are separated by therespective CDR termini as described above. In preferred embodiments theCDRs are as defined by Kabat.

In naturally-occurring antibodies, the six CDRs present on eachmonomeric antibody are short, non-contiguous sequences of amino acidsthat are specifically positioned to form the antigen binding site as theantibody assumes its three dimensional configuration in an aqueousenvironment. The remainder of the heavy and light variable domains showless inter-molecular variability in amino acid sequence and are termedthe framework regions. The framework regions largely adopt a β-sheetconformation and the CDRs form loops which connect, and in some casesform part of, the β-sheet structure. Thus, these framework regions actto form a scaffold that provides for positioning the six CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding site formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto the immunoreactive antigen epitope. The position of CDRs can bereadily identified by one of ordinary skill in the art.

As used herein, the term “fragment” refers to a part or portion of anantibody or antibody chain comprising fewer amino acid residues than anintact or complete antibody or antibody chain. The term “antigen-bindingfragment” refers to a polypeptide fragment of an immunoglobulin orantibody that binds antigen or competes with intact antibody (i.e., withthe intact antibody from which they were derived) for antigen binding(i.e., specific binding to TIGIT). As used herein, the term “fragment”of an antibody molecule includes antigen-binding fragments ofantibodies, for example, an antibody light chain variable domain (VL),an antibody heavy chain variable domain (VH), a single chain antibody(scFv), a F(ab′)2 fragment, a Fab fragment, an Fd fragment, an Fvfragment, and a single domain antibody fragment (DAb). Fragments can beobtained, e.g., via chemical or enzymatic treatment of an intact orcomplete antibody or antibody chain or by recombinant means.

As used herein the term “valency” refers to the number of potentialtarget binding sites in a polypeptide. Each target binding sitespecifically binds one target molecule or specific site on a targetmolecule. When a polypeptide comprises more than one target bindingsite, each target binding site may specifically bind the same ordifferent molecules (e.g., may bind to different ligands or differentantigens, or different epitopes on the same antigen). The subjectbinding molecules have at least one binding site specific for TIGIT.

As used herein, the term “specificity” refers to the ability to bind(e.g., immunoreact with) a given target, e.g., TIGIT. A polypeptide maybe monospecific and contain one or more binding sites which specificallybind a target or a polypeptide may be multispecific and contain two ormore binding sites which specifically bind the same or differenttargets. In one embodiment, an antibody of the invention is specific formore than one target. For example, in one embodiment, a multispecificbinding molecule of the invention binds TIGIT and a second targetmolecule. In this context, the second target molecule is a moleculeother than TIGIT.

As used herein the term “synthetic” with respect to polypeptidesincludes polypeptides which comprise an amino acid sequence that is notnaturally occurring. For example, non-naturally occurring polypeptideswhich are modified forms of naturally occurring polypeptides (e.g.,comprising a mutation such as an addition, substitution or deletion) orwhich comprise a first amino acid sequence (which may or may not benaturally occurring) that is linked in a linear sequence of amino acidsto a second amino acid sequence (which may or may not be naturallyoccurring) to which it is not naturally linked in nature.

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).Preferably, the antibodies of the invention have been engineered toimprove one or more properties, such as antigen binding,stability/half-life or effector function.

As used herein, the term “modified antibody” includes synthetic forms ofantibodies which are altered such that they are not naturally occurring,e.g., antibodies that comprise at least two heavy chain portions but nottwo complete heavy chains (such as, domain deleted antibodies orminibodies); multispecific forms of antibodies (e.g., bispecific,trispecific, etc.) altered to bind to two or more different antigens orto different epitopes on a single antigen; heavy chain molecules joinedto scFv molecules and the like. ScFv molecules are known in the art andare described, e.g., in U.S. Pat. No. 5,892,019. In addition, the term“modified antibody” includes multivalent forms of antibodies (e.g.,trivalent, tetravalent, etc., antibodies that bind to three or morecopies of the same antigen). In another embodiment, a modified antibodyof the invention is a fusion protein comprising at least one heavy chainportion lacking a CH2 domain and comprising a binding domain of apolypeptide comprising the binding portion of one member of a receptorligand pair.

The term “modified antibody” may also be used herein to refer to aminoacid sequence variants of a TIGIT antibody of the invention. It will beunderstood by one of ordinary skill in the art that a TIGIT antibody ofthe invention may be modified to produce a variant TIGIT antibody whichvaries in amino acid sequence in comparison to the TIGIT antibody fromwhich it was derived. For example, nucleotide or amino acidsubstitutions leading to conservative substitutions or changes at“non-essential” amino acid residues may be made (e.g., in CDR and/orframework residues). Amino acid substitutions can include replacement ofone or more amino acids with a naturally occurring or non-natural aminoacid.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable domain thereof. Examples ofantigen binding antibody fragments include Fab, Fab′, F(ab′)2,bi-specific Fab's, and Fv fragments, diabodies, linear antibodies,single-chain antibody molecules, a single chain variable fragment (scFv)and multispecific antibodies formed from antibody fragments (seeHolliger and Hudson, Nature Biotechnol. 23:1126-1136, 2005, the contentsof which are incorporated herein by reference).

As used herein, the term “affinity variant” refers to a variant antibodywhich exhibits one or more changes in amino acid sequence compared to areference TIGIT antibody of the invention, wherein the affinity variantexhibits an altered affinity for TIGIT in comparison to the referenceantibody. Preferably the affinity variant will exhibit improved affinityfor TIGIT, as compared to the reference TIGIT antibody. The improvementmay be apparent as a lower KD for TIGIT, or a slower off-rate for TIGIT.Affinity variants typically exhibit one or more changes in amino acidsequence in the CDRs, as compared to the reference TIGIT antibody. Suchsubstitutions may result in replacement of the original amino acidpresent at a given position in the CDRs with a different amino acidresidue, which may be a naturally occurring amino acid residue or anon-naturally occurring amino acid residue. The amino acid substitutionsmay be conservative or non-conservative.

As used herein, the term “affinity” or “binding affinity” should beunderstood based on the usual meaning in the art in the context ofantibody binding, and reflects the strength and/or stability of bindingbetween an antigen and a binding site on an antibody or antigen bindingfragment thereof.

The anti-TIGIT antibodies provided herein are characterised by highaffinity binding to human TIGIT. Binding affinity for TIGIT may beassessed using standard techniques known to persons of skill in the art.

Binding affinity may also be expressed as the dissociation constant fora particular antibody, or the KD. The lesser the KD value, the strongerthe binding interaction between an antibody and its target antigen. Inone embodiment, binding affinity of a Fab clone comprising a definedVH/VL pairing may be assessed by using methods known in the art, forexample by the ForteBio™ system, by MSD-solution equilibrium titration(SET), or by surface plasmon resonance, e.g. using the Biacore™ systemas described in the accompanying examples. Fab fragments of theantibodies according to the invention typically exhibit a KD for TIGITmeasured by ForteBio™ in the range of from 1×10-10 to 5×10-8 M,optionally 7×10-10 to 4×10-8 M. A KD within this range may be taken asan indication that the Fab, and a corresponding bivalent mAb, exhibithigh affinity binding to hTIGIT. Bivalent mAbs comprising two Fabs that(individually) exhibit KD for hTIGIT within the stated ranges are alsotaken to exhibit high affinity binding to hTIGIT. A MSD KD in the rangeof from 1×10-11 to 5×10-9, optionally 2×10-11 to 1×10-9 may be taken asan indication of high affinity binding to hTIGIT. Fab fragments of theantibodies according to the invention typically exhibit a KD for TIGITmeasured by Biacore™ in the range of from 1×10-10 M to 1×10-9 M,optionally from 1×10-10 to 7×1010, optionally 2×10-10 to 7×10-10 M. A KDwithin this range may be taken as an indication that the Fab, and acorresponding bivalent mAb, exhibit high affinity binding to hTIGIT.

Binding affinity to human TIGIT can also be assessed using a cell-basedsystem as described in the accompanying examples, in which mAbs aretested for binding to mammalian cells (cell lines or ex vivo cells thatexpress TIGIT), for example using ELISA or flow cytometry. High affinityfor TIGIT may be indicated, for example, by an EC50 of no more than 0.5nM by flow cytometric (e.g. FACS) analysis such as that described inExample 10. In certain embodiments, antibodies of the invention exhibita cell binding EC50 of no more than 0.5 nM, optionally no more than 0.2nM. Cell-based determination of affinity expressed as EC50 is preferablydetermined using Jurkat cells expressing hTIGIT or primary CD8 T cellsfrom human peripheral blood mononuclear cells (PBMCs).

As used herein “Treg cells”, or simply “Tregs”, refer to regulatory CD4+T cells—that is, T cells that decrease the effector function(s) ofconventional T cells (CD8 or CD4 T cells). Tregs can be identifiedaccording to methods known in the art, for example using flow cytometryto identify CD4 cells expressing high levels of CD25 and low levels orabsence of CD127.

As summarised above, the invention relates, at least in part, toantibodies, and antigen binding fragments thereof, that bind to TIGIT.The properties and characteristics of the TIGIT antibodies, and antibodyfragments, according to the invention will now be described in furtherdetail.

Anti-TIGIT Antibodies

In one aspect, the present invention provides an isolated antibody orantigen binding fragment thereof which binds to human TIGIT and whichcomprises a heavy chain variable domain comprising a heavy chain CDR1(HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3)selected from the HCDR1, HCDR2 and HCDR3 sequences shown in FIG. 1 andwhich further comprises a light chain variable domain comprising a lightchain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3(LCDR3) selected from the LCDR1, LCDR2, and LCDR3 sequences shown inFIG. 2 . That is, the invention provides an isolated antibody or antigenbinding fragment thereof which binds to human TIGIT and which comprisesa heavy chain variable domain comprising a heavy chain CDR1 (HCDR1), aheavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein:

(i) HCDR1 is selected from the group consisting of SEQ ID Nos: 1, 4, 7,10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 271, 274, and 277;

(ii) HCDR2 is selected from the group consisting of SEQ ID Nos: 2, 5, 8,11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 272, 275, and 278;

(iii) HCDR3 is selected from the group consisting of SEQ ID Nos: 3, 6,9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 273, 276, and 279;

and which further comprises a light chain variable domain comprising alight chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chainCDR3 (LCDR3), wherein

(iv) LCDR1 is selected from the group consisting of SEQ ID Nos: 46, 49,52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 283, 286, and 289;

(v) LCDR2 is selected from the group consisting of SEQ ID Nos: 47, 50,53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 284, 287, and 290;and

(vi) LCDR3 is selected from the group consisting of SEQ ID Nos: 48, 51,54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 285, 288, and 291.

Any given anti-TIGIT antibody or antigen binding fragment thereofcomprising a VH domain paired with a VL domain to form a binding sitefor antigen (human TIGIT) will comprise a combination of 6 CDRs:variable heavy chain CDR3 (HCDR3), variable heavy chain CDR2 (HCDR2),variable heavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3),variable light chain CDR2 (LCDR2) and variable light chain CDR1 (LCDR1).Although many different combinations of 6 CDRs selected from the CDRsequence groups listed above are permissible, and within the scope ofthe invention, certain combinations of 6 CDRs are particularlypreferred; these being the “native” combinations within a single mAbexhibiting high affinity binding to human TIGIT. In certain embodimentsthe antibody or antigen binding fragment comprises a combination ofHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the combination isselected from the group of combinations formed by the HCDRs from eachantibody in FIG. 1 taken with the LCDRs from the corresponding antibodyin FIG. 2 .

That is, in certain embodiments the antibody or antigen binding fragmentcomprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3,wherein the combination is selected from the group consisting of:

(i) HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:2, HCDR3comprising SEQ ID NO:3, LCDR1 comprising SEQ ID NO:46, LCDR2 comprisingSEQ ID NO:47, and LCDR3 comprising SEQ ID NO:48;

(ii) HCDR1 comprising SEQ ID NO:4, HCDR2 comprising SEQ ID NO:5, HCDR3comprising SEQ ID NO:6, LCDR1 comprising SEQ ID NO:49, LCDR2 comprisingSEQ ID NO:50, and LCDR3 comprising SEQ ID NO:51;

(iii) HCDR1 comprising SEQ ID NO:7, HCDR2 comprising SEQ ID NO:8, HCDR3comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO:52, LCDR2 comprisingSEQ ID NO:53, and LCDR3 comprising SEQ ID NO:54;

(iv) HCDR1 comprising SEQ ID NO:10, HCDR2 comprising SEQ ID NO:11, HCDR3comprising SEQ ID NO:12, LCDR1 comprising SEQ ID NO:55, LCDR2 comprisingSEQ ID NO:56, and LCDR3 comprising SEQ ID NO:57;

(v) HCDR1 comprising SEQ ID NO:13, HCDR2 comprising SEQ ID NO:14, HCDR3comprising SEQ ID NO:15, LCDR1 comprising SEQ ID NO:58, LCDR2 comprisingSEQ ID NO:59, and LCDR3 comprising SEQ ID NO:60;

(vi) HCDR1 comprising SEQ ID NO:16, HCDR2 comprising SEQ ID NO:17, HCDR3comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:61, LCDR2 comprisingSEQ ID NO:62, and LCDR3 comprising SEQ ID NO:63;

(vii) HCDR1 comprising SEQ ID NO:19, HCDR2 comprising SEQ ID NO:20,HCDR3 comprising SEQ ID NO:21, LCDR1 comprising SEQ ID NO:64, LCDR2comprising SEQ ID NO:65, and LCDR3 comprising SEQ ID NO:66;

(viii) HCDR1 comprising SEQ ID NO:22, HCDR2 comprising SEQ ID NO:23,HCDR3 comprising SEQ ID NO:24, LCDR1 comprising SEQ ID NO:67, LCDR2comprising SEQ ID NO:68, and LCDR3 comprising SEQ ID NO:69;

(ix) HCDR1 comprising SEQ ID NO:25, HCDR2 comprising SEQ ID NO:26, HCDR3comprising SEQ ID NO:27, LCDR1 comprising SEQ ID NO:70, LCDR2 comprisingSEQ ID NO:71, and LCDR3 comprising SEQ ID NO:72;

(x) HCDR1 comprising SEQ ID NO:28, HCDR2 comprising SEQ ID NO:29, HCDR3comprising SEQ ID NO:30, LCDR1 comprising SEQ ID NO:73, LCDR2 comprisingSEQ ID NO:74, and LCDR3 comprising SEQ ID NO:75;

(xi) HCDR1 comprising SEQ ID NO:31, HCDR2 comprising SEQ ID NO:32, HCDR3comprising SEQ ID NO:33, LCDR1 comprising SEQ ID NO:76, LCDR2 comprisingSEQ ID NO:77, and LCDR3 comprising SEQ ID NO:78;

(xii) HCDR1 comprising SEQ ID NO:34, HCDR2 comprising SEQ ID NO:35,HCDR3 comprising SEQ ID NO:36, LCDR1 comprising SEQ ID NO:79, LCDR2comprising SEQ ID NO:80, and LCDR3 comprising SEQ ID NO:81;

(xiii) HCDR1 comprising SEQ ID NO:37, HCDR2 comprising SEQ ID NO:38,HCDR3 comprising SEQ ID NO:39, LCDR1 comprising SEQ ID NO:82, LCDR2comprising SEQ ID NO:83, and LCDR3 comprising SEQ ID NO:84;

(xiv) HCDR1 comprising SEQ ID NO:40, HCDR2 comprising SEQ ID NO:41,HCDR3 comprising SEQ ID NO:42, LCDR1 comprising SEQ ID NO:85, LCDR2comprising SEQ ID NO:86, and LCDR3 comprising SEQ ID NO:87;

(xv) HCDR1 comprising SEQ ID NO:43, HCDR2 comprising SEQ ID NO:44, HCDR3comprising SEQ ID NO:45, LCDR1 comprising SEQ ID NO:88, LCDR2 comprisingSEQ ID NO:89, and LCDR3 comprising SEQ ID NO:90;

(xvi) HCDR1 comprising SEQ ID NO:271, HCDR2 comprising SEQ ID NO:272,HCDR3 comprising SEQ ID NO:273, LCDR1 comprising SEQ ID NO:283, LCDR2comprising SEQ ID NO:284, and LCDR3 comprising SEQ ID NO:285;

(xvii) HCDR1 comprising SEQ ID NO:274, HCDR2 comprising SEQ ID NO:275,HCDR3 comprising SEQ ID NO:276, LCDR1 comprising SEQ ID NO:286, LCDR2comprising SEQ ID NO:287, and LCDR3 comprising SEQ ID NO:288;

(xviii) HCDR1 comprising SEQ ID NO:277, HCDR2 comprising SEQ ID NO:278,HCDR3 comprising SEQ ID NO:279, LCDR1 comprising SEQ ID NO:289, LCDR2comprising SEQ ID NO:290, and LCDR3 comprising SEQ ID NO:291.

In certain embodiments the antibody or antigen binding fragmentcomprises a heavy chain variable domain having an amino acid sequenceselected from the group consisting of: SEQ ID Nos: 211, 213, 215, 217,219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 327, 329, and 331and amino acid sequences exhibiting at least 90%, 95%, 97%, 98% or 99%sequence identity thereto; and optionally comprising a light chainvariable domain having an amino acid sequence selected from the groupconsisting of: the amino acid sequences of SEQ ID Nos: 212, 214, 216,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 328, 330,and 332 and amino acid sequences exhibiting at least 90%, 95%, 97%, 98%or 99% sequence identity thereto.

Although all possible pairings of VH domains and VL domains selectedfrom the VH and VL domain sequence groups listed above are permissible,and within the scope of the invention, certain combinations VH and VLare particularly preferred; these being the “native” combinations withina single mAb exhibiting high affinity binding to human TIGIT.

In certain embodiments the antibody or antigen binding fragmentcomprises a combination of a heavy chain variable domain and a lightchain variable domain, wherein the combination is selected from thegroup of combinations formed by the VH from each antibody in FIG. 5 , oran amino acid sequence exhibiting at least 90%, 95%, 97%, 98% or 99%sequence identity thereto, taken with the VL from the same antibody inFIG. 5 , or an amino acid sequence exhibiting at least 90%, 95%, 97%,98% or 99% sequence identity thereto. In certain embodiments theantibody or antigen binding fragment comprises a combination of a heavychain variable domain and a light chain variable domain, wherein thecombination is selected from the group consisting of:

(i) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:211 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:212;

(ii) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:213 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:214;

(iii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:215 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO: 216;

(iv) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:217 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:218;

(v) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:219 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:220;

(vi) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:221 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:222;

(vii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:223 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:224;

(viii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:225 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:226;

(ix) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:227 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:228;

(x) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:229 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:230;

(xi) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:231 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:232;

(xii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:233 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:234;

(xiii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:235 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:236;

(xiv) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:237 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:238;

(xv) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:239 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:240;

(xvi) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:327 or an amino acid sequence at least 90% identicalthereto and a light chain variable domain comprising the amino acidsequence of SEQ ID NO:328 or an amino acid sequence at least 90%identical thereto;

(xvii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:329 or an amino acid sequence at least 90% identicalthereto and a light chain variable domain comprising the amino acidsequence of SEQ ID NO:330 or an amino acid sequence at least 90%identical thereto; and

(xviii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:331 or an amino acid sequence at least 90% identicalthereto and a light chain variable domain comprising the amino acidsequence of SEQ ID NO:332 or an amino acid sequence at least 90%identical thereto.

For each of the specific VH/VL combinations listed above, it is alsopermissible, and within the scope of the invention, to combine a VHdomain having an amino acid sequence at least 90%, 92%, 95%, 97% or 99%identical to the recited VH domain sequence with a VL domain having anamino acid sequence at least 90%, 92%, 95%, 97% or 99% identical to therecited VL domain sequence. Embodiments wherein the amino acid sequenceof the VH domain exhibits less than 100% sequence identity with a givenreference VH sequence may nevertheless comprise heavy chain CDRs whichare identical to HCDR1, HCDR2 and HCDR3 of the reference sequence whilstexhibiting amino acid sequence variation within the framework regions.Likewise, embodiments wherein the amino acid sequence of the VL domainexhibits less than 100% sequence identity with a given referencesequence may nevertheless comprise light chain CDRs which are identicalto LCDR1, LCDR2 and LCDR3 of the reference sequence whilst exhibitingamino acid sequence variation within the framework regions.

In the preceding paragraph, and elsewhere herein, the structure of theantibodies/antigen binding fragments is defined on the basis of %sequence identity with a recited reference sequence (with a given SEQ IDNO). In this context, % sequence identity between two amino acidsequences may be determined by comparing these two sequences aligned inan optimum manner and in which the amino acid sequence to be comparedcan comprise additions or deletions with respect to the referencesequence for an optimum alignment between these two sequences. Thepercentage of identity is calculated by determining the number ofidentical positions for which the amino acid residue is identicalbetween the two sequences, by dividing this number of identicalpositions by the total number of positions in the comparison window andby multiplying the result obtained by 100 in order to obtain thepercentage of identity between these two sequences. Typically, thecomparison window with correspond to the full length of the sequencebeing compared. For example, it is possible to use the BLAST program,“BLAST 2 sequences” (Tatusova et al, “Blast 2 sequences—a new tool forcomparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250) available on the sitehttp://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used beingthose given by default (in particular for the parameters “open gappenalty”: 5, and “extension gap penalty”: 2; the matrix chosen being,for example, the matrix “BLOSUM 62” proposed by the program), thepercentage of identity between the two sequences to be compared beingcalculated directly by the program. Determining sequence identity of aquery sequence to a reference sequence is within the ability of theskilled person and can be performed using commercially availableanalysis software such as BLAST™.

In certain preferred embodiments, the antibody or antigen bindingfragment may comprise a heavy chain variable domain and a light chainvariable domain wherein HCDR1 comprises SEQ ID NO: 16, HCDR2 comprisesSEQ ID NO: 17, HCDR3 comprises SEQ ID NO: 18, and LCDR1 comprises SEQ IDNO: 61, LCDR2 comprises SEQ ID NO: 62, and LCDR3 comprises SEQ ID NO:63.

In certain such embodiments, the heavy chain variable domain maycomprise the amino acid sequence shown as SEQ ID NO: 221 or an aminoacid sequence exhibiting at least 90%, 95%, 97%, 98% or 99% sequenceidentity thereto, and the light chain variable domain may comprise theamino acid sequence shown as SEQ ID NO: 222 or an amino acid sequenceexhibiting at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.In certain such embodiments, the heavy chain variable domain and lightchain variable domain are the VH and VL domain of antibody 31282provided herein.

Antibody 31282 provided herein is derived from antibody 29489. Antibody31282 was produced from 29489 by an M-T substitution at amino acid 116in VH FR4 region. This substitution is understood to remove a potentialoxidation site of the antibody and thereby improve stability withoutaffecting function. Antibodies 31282 and 29489 thus share identicalHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences, differing only inthe framework.

Accordingly, in certain embodiments of the antibodies or antigen bindingfragments of the invention, the heavy chain variable domain may comprisethe amino acid sequence shown as SEQ ID NO: 219 or an amino acidsequence exhibiting at least 90%, 95%, 97%, 98% or 99% sequence identitythereto, and the light chain variable domain may comprise the amino acidsequence shown as SEQ ID NO: 220 or an amino acid sequence exhibiting atleast 90%, 95%, 97%, 98% or 99% sequence identity thereto. In certainsuch embodiments, the heavy chain variable domain and light chainvariable domain are the VH and VL domain of antibody 29489 providedherein.

Embodiments wherein the amino acid sequence of the VH domain exhibitsless than 100% sequence identity with the sequence shown as SEQ ID NO:221 or 219 may nevertheless comprise heavy chain CDRs which areidentical to HCDR1, HCDR2 and HCDR3 of SEQ ID NO:221 and 219 (SEQ IDNOs:16, 17 and 18, respectively) whilst exhibiting amino acid sequencevariation within the framework regions. Likewise, embodiments whereinthe amino acid sequence of the VL domain exhibits less than 100%sequence identity with the sequence shown as SEQ ID NO: 222 or 220 maynevertheless comprise light chain CDRs which are identical to LCDR1,LCDR2 and LCDR3 of SEQ ID NO:222 and 220 (SEQ ID NOs:61, 62 and 63,respectively) whilst exhibiting amino acid sequence variation within theframework regions.

Exemplary TIGIT antibodies described herein and having the sequences setout in FIG. 1-5 were developed from 5 parent antibody clones. Table 2summarises the lineage of the antibodies described herein. Naïve parenthuman anti-TIGIT antibodies were expressed in yeast and those exhibitinghigh functional activity against TIGIT were selected (grey rows, named26 . . . ), and underwent affinity maturation. Selected affinity-maturedantibodies then were expressed in mammalian cells (white rows beneatheach parent, named 29 . . . or 3 . . . ). In addition, antibody 31282was produced from 29489 by an M-T substitution at amino acid 116 in VHFR4 region. This substitution is understood to remove a potentialoxidation site of the antibody and thereby improve stability withoutaffecting function. In addition, antibody 31288 was produced from 29494by a V-L substitution at amino acid 2 in VH FR1 region and by an M-Tsubstitution at amino acid 120 in VH FR4 region. The V-L substitution isunderstood to restore the sequence of VH4-39 germline and the M-Tsubstitution to remove a potential oxidation site of the antibody andthereby improve stability without affecting function.

TABLE 2 Antibody VH CDR3 Optimization VH clone Lineage Method Germline26518 26518 Parent VH3-07 29478 26518 H1/H2/H3 VH3-30 26452 26452 ParentVH1-46 29487 26452 H1/H2/H3 VH1-46 29489 26452 H1/H2/H3 VH1-46 3128229489 M116T amino acid VH1-46 mutation 26486 26486 Parent VH4-0B 2949926486 H1/H2/H3 VH4-39 29494 26486 H1/H2/H3 VH4-39 31288 29494 Germlinereversion + VH4-39 M116T amino acid mutation 32919 31288 L1/L2/L3 VH4-3932931 31288 L1/L2/L3 VH4-39 26521 26521 Parent VH1-69 29513 26521H1/H2/H3 VH1-69 26493 26493 Parent VH3-09 29520 26493 H1/H2/H3 VH3-0929523 26493 H1/H2/H3 VH3-33 29527 26493 H1/H2/H3 VH3-30 26432 26432Parent VH1-69 32959 26432 H1/H2/H3 VH1-69

The second generation antibodies exhibit higher affinity than therespective parent antibodies.

In certain embodiments, the invention provides anti-TIGIT antibodies orantigen binding fragments thereof wherein the VH domain is derived froma human V region germline sequence selected from: VH3-07, VH3-30,VH1-46, VH4-0B, VH4-39, VH1-69, VH3-09, VH3-33, VH3-30. In certainpreferred embodiments, the antibody or antigen binding fragment thereofcomprises a VH domain derived from human V region germline VH1-46.

A VH domain is “derived from” a particular V region germline sequence ifthe sequence of the heavy chain variable region is more likely derivedfrom the given germline than from any other.

TIGIT Epitope

The invention also provides an antibody or antigen binding fragmentthereof which binds to human TIGIT at an epitope comprising residues Q56and I109. In certain embodiments, the antibody or antigen bindingfragment thereof binds human TIGIT at least at residues Q56, N58 andI109. In certain embodiments, the antibody or antigen binding fragmentthereof binds human TIGIT at an epitope comprising residues Q56, N58 andI109 and optionally one or more of residues E60, I68, L73 and H76. Incertain embodiments, the antibody or antigen binding fragment thereofbinds human TIGIT at an epitope comprising residues Q56, N58, E60, I68,L73, H76, and I109.

In certain embodiments, the antibody or antigen binding fragment thereofbinds to human TIGIT at an epitope consisting of TIGIT residues Q56,N58, E60, I68, L73, H76, and I109. In certain embodiments, the antibodyor antigen binding fragment thereof binds the same epitope as antibody31282.

Where the antibody or antigen binding fragment binds an epitope of humanTIGIT comprising the indicated TIGIT residues, the antibody binds eachof these residues and optionally other residues of TIGIT. Where theantibody or antigen binding fragment binds an epitope of human TIGITconsisting of TIGIT residues Q56, N58, E60, I68, L73, H76, and I109, theantibody binds each of these residues and no other residues of TIGIT.

An antibody or antigen binding fragment binds to human TIGIT at a givenepitope if it contacts the indicated TIGIT amino acid residue(s) whenbound to TIGIT. As used herein, an antibody contacts a TIGIT residue if,when in the protein complex formed by antibody-TIGIT binding, theresidue meets each of the following criteria: (i) it has a calculatedbinding free energy contribution greater than 0.3 kcal/mol, (ii) it hasan experimental averaged B-factor lower than the mean B-factor of allresidues in the X-ray structure, (iii) it makes at least 3 pairs ofheavy-atom interatomic contacts with antibody atoms at a distance lessthan or equal to 4.0 Angstroms, (iv) it does not make onlysolvent-exposed hydrogen bond or ionic interactions, (v) if it is anon-aromatic polar residue (Asn, Gln, Ser, Thr, Asp, Glu, Lys, or Arg),it makes at least one hydrogen bond or ionic interaction with theantibody. Calculation of binding free energy would be within theabilities of the skilled person. Preferably binding free energy iscalculated using an empirical force field, preferably FoldX. FoldX wouldbe familiar to the skilled person and is publicly available athttp://foldxsuite.crg.eu/. Calculation of binding free energy usingFoldX is also described in Guerois et al. J. Mol. Biol. 2002;320(2):369-87, which is incorporated herein by reference. As would befamiliar to the skilled person, heavy atoms are all non-hydrogen atoms(including C, N, O, S).

Accordingly, the invention also provides an antibody or antigen bindingfragment thereof which contacts human TIGIT at least at residues Q56 andI109. In certain embodiments, the antibody or antigen binding fragmentthereof contacts human TIGIT at least at residues Q56, N58 and I109. Incertain embodiments, the antibody or antigen binding fragment thereofcontacts human TIGIT at least at residues Q56, N58 and I109 andoptionally one or more of residues E60, 168, L73 and H76. In certainembodiments, the antibody or antigen binding fragment thereof contactshuman TIGIT at least at residues Q56, N58, E60, 168 L73, H76, and I109.

In certain such embodiments, the antibody or antigen binding fragmentthereof contacts human TIGIT only at residues Q56, N58, E60, I68, L73,H76, and I109.

Means for determining which residues of TIGIT are contacted by anantibody or antigen-binding fragment are familiar to the skilled person,including X-Ray crystallography, such as that described in Example 23.

Also provided is an isolated antibody or antigen binding fragmentthereof which binds to the same epitope as an antibody orantigen-binding fragment described herein.

Antibody Subtypes

TIGIT antibodies can take various different embodiments in which both aVH domain and a VL domain are present. The term “antibody” herein isused in the broadest sense and encompasses, but is not limited to,monoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), so long as they exhibit the appropriate immunologicalspecificity for a human TIGIT protein. The term “monoclonal antibody” asused herein refers to an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic site. Furthermore, in contrast to conventional (polyclonal)antibody preparations which typically include different antibodiesdirected against different determinants (epitopes) on the antigen, eachmonoclonal antibody is directed against a single determinant or epitopeon the antigen.

In non-limiting embodiments, the TIGIT antibodies provided herein maycomprise CH1 domains and/or CL domains, the amino acid sequence of whichis fully or substantially human. If the TIGIT antibody is intended forhuman therapeutic use, it is typical for the entire constant region ofthe antibody, or at least a part thereof, to have fully or substantiallyhuman amino acid sequence. Therefore, one or more or any combination ofthe CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (andCH4 domain if present) may be fully or substantially human with respectto its amino acid sequence. Such antibodies may be of any human isotype,with human IgG4 and IgG1 being particularly preferred.

Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain andCL domain (and CH4 domain if present) may all have fully orsubstantially human amino acid sequence. In the context of the constantregion of a humanised or chimeric antibody, or an antibody fragment, theterm “substantially human” refers to an amino acid sequence identity ofat least 90%, or at least 92%, or at least 95%, or at least 97%, or atleast 99% with a human constant region. The term “human amino acidsequence” in this context refers to an amino acid sequence which isencoded by a human immunoglobulin gene, which includes germline,rearranged and somatically mutated genes. Such antibodies may be of anyhuman isotype, with human IgG4 and IgG1 being particularly preferred.

Also provided are TIGIT antibodies comprising constant domains of“human” sequence which have been altered, by one or more amino acidadditions, deletions or substitutions with respect to the humansequence.

The TIGIT antibodies provided herein may be of any isotype. Antibodiesintended for human therapeutic use will typically be of the IgA, IgD,IgE IgG, IgM type, often of the IgG type, in which case they can belongto any of the four sub-classes IgG1, IgG2a and b, IgG3 or IgG4. Withineach of these sub-classes it is permitted to make one or more amino acidsubstitutions, insertions or deletions within the Fc portion, or to makeother structural modifications, for example to enhance or reduceFc-dependent functionalities.

In certain preferred embodiments, the TIGIT antibodies provided hereinare IgG antibodies. In certain embodiments, antibodies according to theinvention are IgG1 antibodies. In certain alternate embodiments,antibodies according to the invention are IgG4 antibodies.

IgG4 antibodies are known to undergo Fab arm exchange (FAE), which canresult in unpredictable pharmacodynamic properties of an IgG4 antibody.FAE has been shown to be prevented by the S228P mutation in the hingeregion (Silva et al. J Biol Chem. 2015 Feb. 27; 290(9): 5462-5469).Therefore, in certain such embodiments wherein an antibody according tothe invention is an IgG4 antibody, the antibody comprises the mutationS228P—that is, a serine to proline mutation at position 228 (accordingto EU numbering).

In non-limiting embodiments, it is contemplated that one or more aminoacid substitutions, insertions or deletions may be made within theconstant region of the heavy and/or the light chain, particularly withinthe Fc region. Amino acid substitutions may result in replacement of thesubstituted amino acid with a different naturally occurring amino acid,or with a non-natural or modified amino acid. Other structuralmodifications are also permitted, such as for example changes inglycosylation pattern (e.g. by addition or deletion of N- or O-linkedglycosylation sites). Depending on the intended use of the TIGITantibody, it may be desirable to modify the antibody of the inventionwith respect to its binding properties to Fc receptors, for example tomodulate effector function.

In certain embodiments, the TIGIT antibodies may comprise an Fc regionof a given antibody isotype, for example human IgG1, which is modifiedin order to reduce or substantially eliminate one or more antibodyeffector functions naturally associated with that antibody isotype.

As demonstrated herein, antibodies with cell lytic effector functionscan be effective at reducing Treg cell populations but, surprisingly,without adversely affecting conventional effector T cell populations.This selectivity can allow more potent inhibition of the regulatoryeffect of Tregs whilst retaining anti-tumour effector T cells.

Therefore, in certain alternative embodiments, the TIGIT antibodiesretain one or more of the antibody effector functions naturallyassociated with that antibody isotype. For example, the TIGIT antibodiesof the invention may be IgG1 antibodies that retain ADCC functionality.In further embodiments, the TIGIT antibodies may comprise an Fc regionof a given antibody isotype, for example human IgG1, which is modifiedin order to enhance one or more antibody effector functions naturallyassociated with that antibody isotype. In this context, “antibodyeffector functions” include one or more or all of antibody-dependentcellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)and antibody-dependent cellular phagocytosis (ADCP).

In certain embodiments the anti-TIGIT antibody is a modified antibody.

In certain embodiments is provided a bispecific antibody comprising afirst arm specific for TIGIT and a second arm specific for a secondtarget. In preferred embodiments the second target is an immunecheckpoint molecule. In certain embodiments, the second target is OX40.In certain embodiments, the second target is ICOS. In certainembodiments, the second target is GITR. In certain embodiments, thesecond target is 4-1BB. In certain embodiments, the second target isPD-1. In certain embodiments, the second target is PD-L1. In certainembodiments, the first arm specific of TIGIT comprises a combination ofHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of an antibodyaccording to the invention. In certain embodiments the first armcomprises comprise a heavy chain variable domain and a light chainvariable domain wherein HCDR1 comprises SEQ ID NO: 16, HCDR2 comprisesSEQ ID NO: 17, HCDR3 comprises SEQ ID NO: 18, and LCDR1 comprises SEQ IDNO: 61, LCDR2 comprises SEQ ID NO: 62, and LCDR3 comprises SEQ ID NO:63.

Monoclonal antibodies or antigen-binding fragments thereof that“cross-compete” with the TIGIT antibodies disclosed herein are thosethat bind human TIGIT at site(s) that are identical to, or overlappingwith, the site(s) at which the present TIGIT antibodies bind. Competingmonoclonal antibodies or antigen-binding fragments thereof can beidentified, for example, via an antibody competition assay. For example,a sample of purified or partially purified human TIGIT can be bound to asolid support. Then, an antibody compound or antigen binding fragmentthereof of the present invention and a monoclonal antibody orantigen-binding fragment thereof suspected of being able to compete withsuch invention antibody compound are added. One of the two molecules islabelled. If the labelled compound and the unlabelled compound bind toseparate and discrete sites on TIGIT, the labelled compound will bind tothe same level whether or not the suspected competing compound ispresent. However, if the sites of interaction are identical oroverlapping, the unlabelled compound will compete, and the amount oflabelled compound bound to the antigen will be lowered. If theunlabelled compound is present in excess, very little, if any, labelledcompound will bind.

For purposes of the present invention, competing monoclonal antibodiesor antigen-binding fragments thereof are those that decrease the bindingof the present antibody compounds to TIGIT by about 50%, about 60%,about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%.Details of procedures for carrying out such competition assays are wellknown in the art and can be found, for example, in Harlow and Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988, 567-569, 1988, ISBN 0-87969-314-2. Suchassays can be made quantitative by using purified antibodies. A standardcurve is established by titrating one antibody against itself, i.e., thesame antibody is used for both the label and the competitor. Thecapacity of an unlabelled competing monoclonal antibody orantigen-binding fragment thereof to inhibit the binding of the labelledmolecule to the plate is titrated. The results are plotted, and theconcentrations necessary to achieve the desired degree of bindinginhibition are compared.

Antibodies of the Invention Exhibit High Affinity for Tigit and Competewith CD155

In certain embodiments, the antibodies or antigen binding fragments ofthe invention exhibit high affinity for human TIGIT. In certainembodiments, Fab fragments of the antibodies according to the inventionexhibit a KD for TIGIT measured by ForteBio™ in the range of from1×10-10 to 5×10-8 M, optionally 7×10-10 to 4×10-8 M. In certainembodiments antibodies according to the invention exhibit MSD KD in therange of from 1×10-11 to 5×10-9 M, optionally 2×10-11 to 1×10-9. Incertain embodiments, Fab fragments of the antibodies according to theinvention exhibit a KD for TIGIT measured by Biacore™ in the range offrom 1×10-10 M to 1×10-9 M, optionally 1×10-10 to 7×10-10 M, optionally2×10-10 to 7×10-10 M.

TABLE 3 ForteBio Cell binding Cell binding Fab KD ForteBio ForteBioMSD - Biacore - Jurkat Jurkat Biotinylated Fab KD ForteBio IgG KDmonovalent monovalent Human Mouse Human TIGIT Mouse Fab KD Cyno Human KD(M), KD (M), TIGIT TIGIT HIS (M) TIGIT-Fc (M) TIGIT-Fc (M) TIGIT-Fc (M)human human FON (Fold FON (Fold Clone Monovalent Monovalent MonovalentAvid TIGIT-His TIGIT-His Over Negative) Over Negative) 26518 1.24E−09N.B. 4.47E−09 6.30E−10 154 233 29478 7.03E−10 9.18E−08 1.26E−09 5.27E−10182 500 26452 5.08E−09 N.B. N.B. 4.74E−10 164 47 29487 2.08E−09 N.B.1.55E−07 3.96E−10 161 95 29489 8.81E−10 N.B. 3.52E−08 3.53E−10 1.1E−102.48E−10 162 187 31282 1.34E−09 N.B. 3.77E−08 2.94E−10 26486 2.19E−08N.B. N.B. 5.89E−10 143 199 29499 1.66E−09 2.55E−08 1.45E−08 3.19E−101.9E−11 164 541 29494 1.66E−09 5.36E−08 1.86E−08 3.76E−10 7.0E−112.70E−10 164 511 31288 2.09E−09 2.51E−08 1.92E−10 32919 1.42E−096.57E−09 680 32931 1.18E−09 1.97E−09 741 29499 1.66E−09 2.55E−081.45E−08 3.19E−10 1.9E−11 164 541 26521 9.87E−09 N.B. 1.49E−07 5.41E−10146 218 29513 7.74E−10 8.55E−08 9.56E−09 3.92E−10 2.5E−11 156 406 264934.06E−08 2.67E−08 N.B. 1.49E−09 80 463 29520 1.31E−09 1.95E−09 2.68E−093.84E−10 2.1E−10 7.16E−10 166 535 29523 3.84E−09 1.89E−08 2.79E−085.31E−10 1.7E−09 150 502 29527 1.33E−09 2.02E−08 1.76E−08 3.50E−106.4E−10 142 414 26432 1.31E−08 N.B. N.B. 4.62E−09

As demonstrated in the Examples, antibody 31282 exhibits surprisinglyhigh affinity for TIGIT expressed on transgenic cells. Accordingly, incertain embodiments, an anti-TIGIT antibody or antigen binding fragmentprovided herein exhibits a binding EC50 for human TIGIT of less than 0.5nM. In preferred such embodiments, the antibody or antigen bindingfragment exhibits a binding EC50 of from about 0.05 to about 0.4 nM,preferably from about 0.05 to about 0.3 nM, preferably from about 0.05to about 0.2 nM, preferably from about 0.05 to about 0.15 nM. In certainpreferred embodiments, the antibody or antigen binding fragment exhibitsa binding EC50 for human TIGIT of about 0.1 nM. In preferredembodiments, the antibody comprises the CDRs of antibody 31282.Preferably the EC50 is determined using Jurkat cells expressing humanTIGIT, as described in Example 18. In certain embodiments, antibodies orantigen binding fragments of the invention cross-react with mouse TIGITand/or cynomolgus TIGIT.

Since the “29 . . . ” second generation antibodies are affinity maturedprogeny of the highly functional parent antibodies, it is expected thatthey will exhibit at least similar or equivalent functional propertiesas the parent antibodies, and vice versa.

As described herein, in certain embodiments an antibody or antigenbinding fragment of the invention has equivalent affinity for TIGITexpressed by CD8 T cells and expressed by Treg cells. As used herein, anantibody or antigen binding fragment has “equivalent affinity” for CD8 Tcells and Treg cells if the affinity for CD8 T cells is in the range of0.5-1.5 times that of the affinity for Treg cells. For example, anantibody having equivalent affinity for CD8 T cells and Treg cells whichexhibits an affinity for Treg cells of 0.03 nM would exhibit an affinityfor CD8 T cells in the range of 0.015-0.045 nM.

Table 3 provides a summary of the affinity properties of the anti-TIGITantibodies of the invention, with grey cells indicating parent antibodyclones, with second and third generation antibodies of each lineageshown immediately below the respective parent antibody (see also Table2).

As demonstrated in the Examples, antibody 31282 exhibits surprisinglyhigh affinity for TIGIT expressed on human primary CD8+ T cells.Accordingly, in certain embodiments, an anti-TIGIT antibody or antigenbinding fragment provided herein exhibits a binding EC50 for human TIGITof less than 0.5 nM. In preferred such embodiments, the antibody orantigen binding fragment exhibits a binding EC50 of from about 0.05 toabout 0.4 nM, preferably from about 0.1 to about 0.3 nM. In certainpreferred embodiments, the antibody or antigen binding fragment exhibitsa binding EC50 for human TIGIT of about 0.2 nM. In preferredembodiments, the antibody or antigen binding fragment comprises the CDRsof antibody 31282. Preferably the EC50 is determined using CD8+ T cellsfrom human PBMCs, preferably from a healthy individual, as described inExample 18.

As demonstrated in the accompanying examples, in certain embodimentsantibodies or antigen binding fragment of the invention exhibit highaffinity for TIGIT-expressing CD8 T cells and high affinity forTIGIT-expressing Treg cells. In certain embodiments, antibodies orantigen binding fragment of the invention exhibit an affinity forTIGIT-expressing CD8 T cells and TIGIT-expressing Treg cellscharacterised by an EC50 less than 0.5 nM, preferably less than 0.3 nM,preferably less than 0.2 nM. In certain embodiments, the antibodies orantigen binding fragment of the invention exhibit equivalent affinityfor TIGIT-expressing CD8 T cells and for TIGIT-expressing Treg cells.

Antibodies according to the invention (e.g. antibody 31282) exhibitsurprisingly high affinity for CD8+ T cells from cancer patients. Thisis particularly advantageous, since increasing effector activity of Tcells from cancer patients by inhibition of TIGIT signalling can lead tomore effective tumour control. Accordingly, in certain embodiments, ananti-TIGIT antibody or antigen binding fragment provided herein exhibitsa binding EC50 of less than 0.5 nM for human TIGIT on human CD8+ T cellsfrom cancer patients. In preferred such embodiments, the antibody orantigen binding fragment exhibits a binding EC50 of from about 0.05 toabout 0.4 nM, preferably from about 0.1 to about 0.3 nM. In certainpreferred embodiments, the antibody or antigen binding fragment exhibitsan EC50 for human TIGIT of from about 0.1 nM to about 0.2 nM. Inpreferred embodiments, the antibody or antigen binding fragmentcomprises the CDRs of antibody 31282. Preferably the EC50 is determinedusing CD8+ T cells from PBMCs taken from a patient with cancer, asdescribed in Example 18.

As demonstrated in the accompanying Examples, in certain embodimentsantibodies or antigen binding fragments of the invention compete withCD155/PVR for TIGIT binding. In certain embodiments, an antibody orantigen binding fragment of the invention exhibits competition withCD155 characterised by an IC50 of 0.2 nM or less, preferably 0.1 nM orless. In certain embodiments, the antibody or antigen binding fragmentexhibits competition with CD155 characterised by an IC50 of about 0.05nM or less. In certain preferred embodiments, the exhibited IC50 isabout 0.05 nM. Without wishing to be bound by theory, competition ofantibodies with CD155 for TIGIT binding is expected to decrease levelsof CD155-induced TIGIT-mediated signalling, thereby increasing levels ofeffector T cell activation.

The invention further provides “affinity variants” of the antibodiesdescribed herein.

The invention also provides an isolated antibody or antigen bindingfragment thereof which cross-competes for binding to human TIGIT with anantibody or antigen-binding fragment described herein.

Antibodies of the Invention Promote Pro-Inflammatory T Cell Activity

Antibodies according to the invention (e.g. antibody 31282) aresurprisingly effective at promoting pro-inflammatory activity of CD8+ Tcells. As demonstrated in the Examples, antibodies or antigen bindingfragments according to the invention (especially 31282) are moreeffective at promoting pro-inflammatory CD8+ T cell activity (indicatedby IFNg release) than comparator anti-TIGIT antibodies (see FIG. 24 ).This improved efficacy versus comparator antibodies was demonstrated inTIGIT-expressing transgenic Jurkat reporter cells and in primary CD8 Tcells. Accordingly, in certain embodiments, an anti-TIGIT antibody orantigen binding fragment provided herein exhibits an activation EC50 ofless than 5 nM for human TIGIT expressed by Jurkat reporter cells asdescribed in Example 19. In preferred such embodiments, the antibody orantigen binding fragment exhibits an EC50 of from about 1 nM to about 4nM, preferably from about 2 nM to about 4 nM.

In certain embodiments, an anti-TIGIT antibody or antigen bindingfragment provided herein exhibits an activation EC50 of less than 0.4 nMfor CD8 T cells from healthy individuals as described in Example 19. CD8T cell activity (i.e. pro-inflammatory activity) may be measured byinflammatory cytokine (e.g. IFNg) production. In preferred suchembodiments, the antibody or antigen binding fragment exhibits an EC50of from about 0.05 nM to about 0.4 nM, preferably from about 0.1 nM toabout 0.2 nM. Preferably the EC50 is determined using CD8+ T cells fromPBMCs taken from a healthy individual, as described in Example 19.

It is additionally and surprisingly demonstrated in the accompanyingExamples that the provided anti-TIGIT antibodies are effective atincreasing the activity of gamma-delta (γδ, or g/d) T cells (i.e. Tcells expressing the γδ TCR subunits, as opposed to the conventional αβTCR subunits). Such γδ T cells form a distinct and important componentof the immune system and the ability of the antibodies provided hereinto promote activity of these cells highlights the utility of theantibodies.

Accordingly, also provided herein is a method of promoting γδ T cellactivity comprising contacting a population of γδ T cells with ananti-TIGIT antibody. In certain embodiments the method is performed invitro. In certain embodiments the method is performed in vivo in a humansubject. In certain such embodiments the human subject has cancer. Incertain embodiments the anti-TIGIT antibody or antigen binding fragmentcomprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3sequences of an antibody according to the invention. In certainembodiments the anti-TIGIT antibody comprises a heavy chain variabledomain and a light chain variable domain wherein HCDR1 comprises SEQ IDNO: 16, HCDR2 comprises SEQ ID NO: 17, HCDR3 comprises SEQ ID NO: 18,and LCDR1 comprises SEQ ID NO: 61, LCDR2 comprises SEQ ID NO: 62, andLCDR3 comprises SEQ ID NO: 63.

Selective Depletion of T-reg Cells

As demonstrated herein, anti-TIGIT antibodies are able to selectivelydeplete TIGIT-expressing Treg cells. That is, anti-TIGIT antibodiesreduce the proportion of TIGIT-expressing Treg cells relative to thetotal population of T cells to a greater extent than they reduce theproportion of effector or memory CD4 or CD8 T cells.

In certain embodiments, the antibody or antigen binding fragment thereofselectively depletes TIGIT-expressing Treg cells.

This selective depletion of TIGIT-expressing Treg cells can be mediatedvia selective lysis of the TIGIT-expressing Tregs (e.g. by ADCC or CDC(see FIGS. 20, 21, and 25 ). TIGIT-expressing Tregs are understood to bethe more potent regulatory cells than Tregs not expressing TIGIT.Without wishing to be bound by theory, selective depletion by lysis ofTIGIT-expressing Treg cells is expected to increase T cell effectorfunction (e.g. T-cell mediated cytotoxicity, pro-inflammatory cytokinerelease) by depleting the overall number of Treg cells but alsodepleting those Treg cells exhibiting the more potent regulatoryfunction. This increased T cell effector function is demonstrated inFIG. 24 .

Therefore, in certain embodiments, antibodies or antigen bindingfragments of the invention selectively lyse TIGIT-expressing Treg cells.

Selective depletion of Treg cells expressing TIGIT can also be mediatedby inducing internalisation of the TIGIT receptor such that it is nolonger expressed at the cell membrane. Without wishing to be bound bytheory, by inducing TIGIT internalisation such that TIGIT+ Treg cellsbecome TIGIT− Treg cells, the regulatory function of these cells isexpected to become less potent (since TIGIT+ Tregs are more potentregulatory cells). As a result of the receptor internalisation andsubsequent drop in regulatory potency of these Tregs, T cell effectorfunction is expected to increase. Therefore, in certain embodiments,antibodies or antigen binding fragments of the invention inhibitsuppressive activity of TIGIT-expressing Treg cells, preferably byinducing internalisation of TIGIT by TIGIT-expressing Treg cells.

It is particularly advantageous for anti-TIGIT antibodies according tothe invention to exhibit high affinity for CD8 T cells and Treg cellsand also to exhibit selective depletion of Treg cells, thereby promotingT cell effector function via two mechanisms. Retention of antibodyeffector function (e.g. ADCC, CDC) results in effective depletion of theTregs and the selectivity means the antibody effector function does notresult in unwanted depletion of effector T cells. The selectivity isparticularly surprising since previous attempts to produce an anti-TIGITantibody have sought to eliminate antibody effector function in order toavoid lysis of effector T cells expressing TIGIT. Moreover, becauseTIGIT antibodies of the invention exhibit affinity for effector T cells(e.g. CD8 T cells), TIGIT-mediated signalling in these cells can beinhibited by competition for CD155 binding and/or inducinginternalisation of TIGIT on effector T cells. In combination, theseeffects of the antibodies of the invention can result in significantupregulation of T cell effector function.

Further surprising advantageous properties exhibited by antibodies andantigen binding fragments according to the invention include increasingthe T cell effector function (e.g. release of proinflammatory cytokines)of tumour infiltrating lymphocytes (TILs). Exposure to the tumourmicroenvironment can lead to TILs exhibiting anergic or so-called“exhausted” phenotypes, possibly due to antigen over-exposure and/or animmunosuppressive tumour microenvironment. Enhancing the effectorfunction of TILs is desirable as it is these cells that are infiltratingthe tumour itself and thus positioned at a locus best-suited to reducetumour size or growth; however due to the anergic or exhausted phenotypeof many TILs, it is expected to be difficult to potentiate theireffector function. The increase in proinflammatory response from TILsfollowing exposure to antibodies of the invention is thereforesurprising and indicates the antibodies may be particularly effectivetherapeutic agents.

Still further surprising advantageous properties exhibited by antibodiesand antigen binding fragments include the ability to increase thepro-inflammatory activity of gamma-delta (γδ) T cells. The ability topromote activity of non-conventional T cells such as γδ T cells has notpreviously been reported for an anti-TIGIT antibody and offers thepotential to treat diseases other than cancer in which γδ T cells areknown to be important. For example, γδ T cells have been reported to beinvolved in the response to pathogenic infection (bacterial, viral (e.g.CMV), fungal) as well as to have a role in protecting from autoimmunediseases. In addition, the surprising ability to promote activity ofnon-conventional T cells provides further potency to the anti-tumoureffects of the antibodies.

In a further aspect is provided a method for selectively depleting Tregcells from a population of T cells, comprising contacting the populationof T cells with an anti-TIGIT antibody or antigen binding fragmentthereof, whereby the anti-TIGIT antibody selectively depletes thepopulation of Treg cells. In certain embodiments the method is performedin vitro. In certain embodiments the method is performed in vivo in ahuman subject. In certain such embodiments the human subject has cancer.In certain embodiments the anti-TIGIT antibody or antigen bindingfragment comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2and LCDR3 sequences of an antibody according to the invention. Incertain embodiments the anti-TIGIT antibody comprises a heavy chainvariable domain and a light chain variable domain wherein HCDR1comprises SEQ ID NO: 16, HCDR2 comprises SEQ ID NO: 17, HCDR3 comprisesSEQ ID NO: 18, and LCDR1 comprises SEQ ID NO: 61, LCDR2 comprises SEQ IDNO: 62, and LCDR3 comprises SEQ ID NO: 63.

As demonstrated in the accompanying examples, the present invention alsoprovides anti-TIGIT antibodies that do not compete with CD155/PVR forTIGIT binding. Therefore, in a further aspect, the invention provides ahuman TIGIT antibody or antigen binding fragment thereof that does notcompete with CD155/PVR for human TIGIT binding. In certain suchembodiments, Fab fragments of the CD155 non-competitive anti-TIGITantibodies according to the invention exhibit a KD for TIGIT measured byForteBio™ in the range of from 5×10-9 to 5×10-8 M, optionally 1×10-8 to3×10-8 M.

In certain preferred embodiments, the antibody may comprise a heavychain variable domain and a light chain variable domain wherein HCDR1comprises SEQ ID NO: 280, HCDR2 comprises SEQ ID NO: 281, HCDR3comprises SEQ ID NO: 282, and LCDR1 comprises SEQ ID NO: 292, LCDR2comprises SEQ ID NO: 293, and LCDR3 comprises SEQ ID NO: 294. In certainsuch embodiments, the heavy chain variable domain may comprise the aminoacid sequence shown as SEQ ID NO: 333 or an amino acid sequenceexhibiting at least 90%, 95%, 97%, 98% or 99% sequence identity thereto,and the light chain variable domain may comprise the amino acid sequenceshown as SEQ ID NO: 334 or an amino acid sequence exhibiting at least90%, 95%, 97%, 98% or 99% sequence identity thereto.

Embodiments wherein the amino acid sequence of the VH domain exhibitsless than 100% sequence identity with the sequence shown as SEQ ID NO:333 may nevertheless comprise heavy chain CDRs which are identical toHCDR1, HCDR2 and HCDR3 of SEQ ID NO:333 (SEQ ID NOs:280, 281 and 282,respectively) whilst exhibiting amino acid sequence variation within theframework regions. Likewise, embodiments wherein the amino acid sequenceof the VL domain exhibits less than 100% sequence identity with thesequence shown as SEQ ID NO: 334 may nevertheless comprise light chainCDRs which are identical to LCDR1, LCDR2 and LCDR3 of SEQ ID NO:334 (SEQID NOs:292, 293 and 294, respectively) whilst exhibiting amino acidsequence variation within the framework regions.

In certain preferred embodiments, the antibody may comprise a heavychain variable domain and a light chain variable domain wherein HCDR1comprises SEQ ID NO: 353, HCDR2 comprises SEQ ID NO: 354, HCDR3comprises SEQ ID NO: 355, and LCDR1 comprises SEQ ID NO: 356, LCDR2comprises SEQ ID NO: 357, and LCDR3 comprises SEQ ID NO: 358. In certainsuch embodiments, the heavy chain variable domain may comprise the aminoacid sequence shown as SEQ ID NO: 367 or an amino acid sequenceexhibiting at least 90%, 95%, 97%, 98% or 99% sequence identity thereto,and the light chain variable domain may comprise the amino acid sequenceshown as SEQ ID NO: 368 or an amino acid sequence exhibiting at least90%, 95%, 97%, 98% or 99% sequence identity thereto.

Embodiments wherein the amino acid sequence of the VH domain exhibitsless than 100% sequence identity with the sequence shown as SEQ ID NO:367 may nevertheless comprise heavy chain CDRs which are identical toHCDR1, HCDR2 and HCDR3 of SEQ ID NO:367 (SEQ ID NOs:353, 354 and 355,respectively) whilst exhibiting amino acid sequence variation within theframework regions. Likewise, embodiments wherein the amino acid sequenceof the VL domain exhibits less than 100% sequence identity with thesequence shown as SEQ ID NO: 368 may nevertheless comprise light chainCDRs which are identical to LCDR1, LCDR2 and LCDR3 of SEQ ID NO:368 (SEQID NOs:356, 357 and 358, respectively) whilst exhibiting amino acidsequence variation within the framework regions.

Polynucleotides, Vectors and Expression Systems

The invention also provides polynucleotide molecules encoding the TIGITantibodies of the invention, also expression vectors containing anucleotide sequences which encode the TIGIT antibodies of the inventionoperably linked to regulatory sequences which permit expression of theantigen binding polypeptide in a host cell or cell-free expressionsystem, and a host cell or cell-free expression system containing thisexpression vector.

Polynucleotide molecules encoding the TIGIT antibodies of the inventioninclude, for example, recombinant DNA molecules. The terms “nucleicacid”, “polynucleotide” or a “polynucleotide molecule” as used hereininterchangeably and refer to any DNA or RNA molecule, either single- ordouble-stranded and, if single-stranded, the molecule of itscomplementary sequence. In discussing nucleic acid molecules, a sequenceor structure of a particular nucleic acid molecule may be describedherein according to the normal convention of providing the sequence inthe 5′ to 3′ direction. In some embodiments of the invention, nucleicacids or polynucleotides are “isolated”. This term, when applied to anucleic acid molecule, refers to a nucleic acid molecule that isseparated from sequences with which it is immediately contiguous in thenaturally occurring genome of the organism in which it originated. Forexample, an “isolated nucleic acid” may comprise a DNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryotic or eukaryotic cell or non-human hostorganism. When applied to RNA, the term “isolated polynucleotide” refersprimarily to an RNA molecule encoded by an isolated DNA molecule asdefined above. Alternatively, the term may refer to an RNA molecule thathas been purified/separated from other nucleic acids with which it wouldbe associated in its natural state (i.e., in cells or tissues). Anisolated polynucleotide (either DNA or RNA) may further represent amolecule produced directly by biological or synthetic means andseparated from other components present during its production.

For recombinant production of a TIGIT antibody according to theinvention, a recombinant polynucleotide encoding it may be prepared(using standard molecular biology techniques) and inserted into areplicable vector for expression in a chosen host cell, or a cell-freeexpression system. Suitable host cells may be prokaryote, yeast, orhigher eukaryote cells, specifically mammalian cells. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen.Virol. 36:59-74, 1977); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216, 1980; or CHO derived clones like CHO-K1, ATCC CCL-61,Kao and Puck, Genetics of somatic mammalian cells, VII. Induction andisolation of nutritional mutants in Chinese hamster cells, Proc. Natl.Acad. Sci. 60:1275-1281, 1968); mouse sertoli cells (TM4; Mather, Biol.Reprod. 23:243-252, 1980); mouse myeloma cells SP2/0-AG14 (ATCC CRL1581; ATCC CRL 8287) or NSO (HPA culture collections no. 85110503);monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumour (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68,1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), aswell as DSM's PERC-6 cell line. Expression vectors suitable for use ineach of these host cells are also generally known in the art.

It should be noted that the term “host cell” generally refers to acultured cell line. Whole human beings into which an expression vectorencoding an antigen binding polypeptide according to the invention hasbeen introduced are explicitly excluded from the definition of a “hostcell”.

In an important aspect, the invention also provides a method ofproducing a TIGIT antibody of the invention which comprises culturing ahost cell (or cell free expression system) containing polynucleotide(e.g. an expression vector) encoding the TIGIT antibody under conditionswhich permit expression of the TIGIT antibody, and recovering theexpressed TIGIT antibody. This recombinant expression process can beused for large scale production of TIGIT antibodies according to theinvention, including monoclonal antibodies intended for humantherapeutic use. Suitable vectors, cell lines and production processesfor large scale manufacture of recombinant antibodies suitable for invivo therapeutic use are generally available in the art and will be wellknown to the skilled person.

Therefore, in accordance with the invention is provided an isolatedpolynucleotide or combination of isolated polynucleotides encoding anantibody or antigen binding fragment comprising a combination of HCDR1,HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the combination isselected from the group consisting of:

(i) HCDR1 comprising SEQ ID NO: 16, HCDR2 comprising SEQ ID NO: 17,HCDR3 comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:61, LCDR2comprising SEQ ID NO:62, and LCDR3 comprising SEQ ID NO:63;

(ii) HCDR1 comprising SEQ ID NO:4, HCDR2 comprising SEQ ID NO:5, HCDR3comprising SEQ ID NO:6, LCDR1 comprising SEQ ID NO:49, LCDR2 comprisingSEQ ID NO:50, and LCDR3 comprising SEQ ID NO:51;

(iii) HCDR1 comprising SEQ ID NO:7, HCDR2 comprising SEQ ID NO:8, HCDR3comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO:52, LCDR2 comprisingSEQ ID NO:53, and LCDR3 comprising SEQ ID NO:54;

(iv) HCDR1 comprising SEQ ID NO:10, HCDR2 comprising SEQ ID NO:11, HCDR3comprising SEQ ID NO:12, LCDR1 comprising SEQ ID NO:55, LCDR2 comprisingSEQ ID NO:56, and LCDR3 comprising SEQ ID NO:57;

(v) HCDR1 comprising SEQ ID NO:13, HCDR2 comprising SEQ ID NO:14, HCDR3comprising SEQ ID NO:15, LCDR1 comprising SEQ ID NO:58, LCDR2 comprisingSEQ ID NO:59, and LCDR3 comprising SEQ ID NO:60;

(vi) HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:2, HCDR3comprising SEQ ID NO:3, LCDR1 comprising SEQ ID NO:46, LCDR2 comprisingSEQ ID NO:47, and LCDR3 comprising SEQ ID NO:48;

(vii) HCDR1 comprising SEQ ID NO:19, HCDR2 comprising SEQ ID NO:20,HCDR3 comprising SEQ ID NO:21, LCDR1 comprising SEQ ID NO:64, LCDR2comprising SEQ ID NO:65, and LCDR3 comprising SEQ ID NO:66;

(viii) HCDR1 comprising SEQ ID NO:22, HCDR2 comprising SEQ ID NO:23,HCDR3 comprising SEQ ID NO:24, LCDR1 comprising SEQ ID NO:67, LCDR2comprising SEQ ID NO:68, and LCDR3 comprising SEQ ID NO:69;

(ix) HCDR1 comprising SEQ ID NO:25, HCDR2 comprising SEQ ID NO:26, HCDR3comprising SEQ ID NO:27, LCDR1 comprising SEQ ID NO:70, LCDR2 comprisingSEQ ID NO:71, and LCDR3 comprising SEQ ID NO:72;

(x) HCDR1 comprising SEQ ID NO:28, HCDR2 comprising SEQ ID NO:29, HCDR3comprising SEQ ID NO:30, LCDR1 comprising SEQ ID NO:73, LCDR2 comprisingSEQ ID NO:74, and LCDR3 comprising SEQ ID NO:75;

(xi) HCDR1 comprising SEQ ID NO:31, HCDR2 comprising SEQ ID NO:32, HCDR3comprising SEQ ID NO:33, LCDR1 comprising SEQ ID NO:76, LCDR2 comprisingSEQ ID NO:77, and LCDR3 comprising SEQ ID NO:78;

(xii) HCDR1 comprising SEQ ID NO:34, HCDR2 comprising SEQ ID NO:35,HCDR3 comprising SEQ ID NO:36, LCDR1 comprising SEQ ID NO:79, LCDR2comprising SEQ ID NO:80, and LCDR3 comprising SEQ ID NO:81;

(xiii) HCDR1 comprising SEQ ID NO:37, HCDR2 comprising SEQ ID NO:38,HCDR3 comprising SEQ ID NO:39, LCDR1 comprising SEQ ID NO:82, LCDR2comprising SEQ ID NO:83, and LCDR3 comprising SEQ ID NO:84;

(xiv) HCDR1 comprising SEQ ID NO:40, HCDR2 comprising SEQ ID NO:41,HCDR3 comprising SEQ ID NO:42, LCDR1 comprising SEQ ID NO:85, LCDR2comprising SEQ ID NO:86, and LCDR3 comprising SEQ ID NO:87;

(xv) HCDR1 comprising SEQ ID NO:43, HCDR2 comprising SEQ ID NO:44, HCDR3comprising SEQ ID NO:45, LCDR1 comprising SEQ ID NO:88, LCDR2 comprisingSEQ ID NO:89, and LCDR3 comprising SEQ ID NO:90;

(xvi) HCDR1 comprising SEQ ID NO:271, HCDR2 comprising SEQ ID NO:272,HCDR3 comprising SEQ ID NO:273, LCDR1 comprising SEQ ID NO:283, LCDR2comprising SEQ ID NO:284, and LCDR3 comprising SEQ ID NO:285;

(xvii) HCDR1 comprising SEQ ID NO:274, HCDR2 comprising SEQ ID NO:275,HCDR3 comprising SEQ ID NO:276, LCDR1 comprising SEQ ID NO:286, LCDR2comprising SEQ ID NO:287, and LCDR3 comprising SEQ ID NO:288;

(xviii) HCDR1 comprising SEQ ID NO:277, HCDR2 comprising SEQ ID NO:278,HCDR3 comprising SEQ ID NO:279, LCDR1 comprising SEQ ID NO:289, LCDR2comprising SEQ ID NO:290, and LCDR3 comprising SEQ ID NO:291.

In certain embodiments is provided an isolated polynucleotide orcombination of isolated polynucleotides encoding an antibody or antigenbinding fragment comprising a combination of HCDR1, HCDR2, HCDR3, LCDR1,LCDR2 and LCDR3 wherein:

(i) HCDR1 comprises or consists of SEQ ID NO: 16, HCDR2 comprises orconsists of SEQ ID NO: 17, HCDR3 comprises or consists of SEQ ID NO:18,LCDR1 comprises or consists of SEQ ID NO:61, LCDR2 comprises or consistsof SEQ ID NO:62, and LCDR3 comprises or consists of SEQ ID NO:63.

Also in accordance with the invention there is provided an isolatedpolynucleotide or combination of isolated polynucleotides encoding anantibody or antigen binding fragment described herein. In certainembodiments is provided an isolated polynucleotide encoding antibody31282 provided herein, or an antigen binding fragment thereof.

Also, in accordance with the invention there is provided an isolatedpolynucleotide encoding a VH and/or a VL domain of an anti-TIGITantibody, wherein the polynucleotide comprises one or more sequencesselected from the group consisting of SEQ ID Nos: 241-270, 335-342 and369-370. In certain embodiments, the isolated polynucleotide comprises asequence according to SEQ ID NO: 251 and/or a sequence according to SEQID NO: 252. In certain embodiments where the polynucleotide comprises asequence according to SEQ ID NO: 251 and a sequence according to SEQ IDNO: 252, the sequences are contiguous. In certain embodiments where thepolynucleotide comprises a sequence according to SEQ ID NO: 251 and asequence according to SEQ ID NO: 252, the sequences are not contiguous.

Also, in accordance with the invention there is provided an expressionvector comprising a polynucleotide according to the invention operablylinked to regulatory sequences which permit expression of the antigenbinding polypeptide in a host cell or cell-free expression system.

Also, in accordance with the invention there is provided a host cell orcell-free expression system containing an expression vector according tothe invention.

Also, in accordance with the invention there is provided a method ofproducing a recombinant antibody or antigen binding fragment thereofwhich comprises culturing the host cell or cell free expression systemaccording to the invention under conditions which permit expression ofthe antibody or antigen binding fragment and recovering the expressedantibody or antigen binding fragment.

Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions comprising anantibody or antigen binding fragment according to the inventionformulated with one or more a pharmaceutically acceptable carriers orexcipients. Such compositions may include one or a combination of (e.g.,two or more different) TIGIT antibodies. Techniques for formulatingantibodies for human therapeutic use are well known in the art and arereviewed, for example, in Wang et al., Journal of PharmaceuticalSciences, Vol. 96, pp 1-26, 2007.

The TIGIT antibodies and pharmaceutical compositions provided hereinhave utility in therapy, in particular the therapeutic treatment ofdisease, in particular conditions that benefit from inhibition of TIGITfunction.

Combination Products

As demonstrated herein, the antibodies of the invention or antigenbinding fragments thereof are particularly effective when administeredin combination with immune checkpoint inhibitors—specifically anti-ICOSantagonist antibodies or anti-PD-1 antibodies (that is, antagonistantibodies specific for human immunoregulatory molecule PD-1).Administration of anti-TIGIT antibodies in combination with an anti-ICOSor anti-PD-1 antibody results in a synergistic reduction in tumourgrowth compared to either antibody alone. Similar effects are expectedto be observed using a combination of an anti-TIGIT antibody accordingto the invention and an anti-PD-L1 antibody.

It is further demonstrated herein that antibodies of the invention orantigen binding fragments thereof are particularly effective whenadministered in combination with an agonist antibody specific to animmune checkpoint co-stimulatory molecule—specifically anti-4-1BB,anti-OX40 or anti-GITR agonist antibodies. Administration of anti-TIGITantibodies in combination with an anti-4-1BB, anti-OX40 or anti-GITRagonist antibody results in a synergistic reduction in tumour growthcompared to either antibody alone.

In a further aspect is provided a combination product comprising ananti-TIGIT antibody or antigen binding fragment thereof and one or moreof a chemotherapeutic agent, an anti-PD1 antibody, an anti-PD-L1antibody, an anti-41BB antibody, an anti-OX40 antibody, an anti-GITRantibody, and an anti-ICOS antibody. In certain preferred embodiments,the anti-TIGIT antibody or antigen binding fragment is an antibody orantigen binding fragment provided in accordance with the invention. In amost preferred embodiment, the anti-TIGIT antibody or antigen bindingfragment comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2and LCDR3, wherein:

HCDR1 comprises SEQ ID NO: 16 (YTFTSYYMH), HCDR2 comprises SEQ ID NO: 17(VIGPSGASTSYAQKFQG), HCDR3 comprises SEQ ID NO: 18 (ARDHSDYWSGIMEV),LCDR1 comprises SEQ ID NO: 61 (RASQSVRSSYLA), LCDR2 comprisesSEQ ID NO: 62 (GASSRAT),  and LCDR3 comprises SEQ ID NO: 63 (QQYFSPPWT).

Also provided is a combination as provided herein for use in a method oftreating cancer or viral infection, optionally wherein the viralinfection is CMV infection. Further provided is a combination asprovided herein for use in a method provided herein.

As used herein, where two or more active agents are provided as a“combination”, “therapeutic combination” or “combination therapy” (theterms are used interchangeably), this does not require or exclude thatthe active agents are formulated into a single composition. Acombination therapy is given its conventional interpretation of two ormore active agents to be administered such that the patient can derive abenefit from each agent. “Combination therapy” does not necessitateco-formulation, co-administration, simultaneous administration or fixeddose formulation.

Therapeutic Methods

The TIGIT antibodies, or antigen binding fragments thereof andpharmaceutical compositions provided herein can be used to inhibit thegrowth of cancerous tumour cells in vivo and are therefore useful in thetreatment of tumours.

Accordingly, further aspects of the invention relate to methods ofinhibiting tumour cell growth in a human patient, and also methods oftreating or preventing cancer, which comprise administering to a patientin need thereof an effective amount of a TIGIT antibody or antigenbinding fragment as described herein, a pharmaceutical composition asdescribed herein, or a combination as described herein.

Another aspect of the invention provides a TIGIT antibody or antigenbinding fragment as described herein for use in inhibiting the growth oftumour cells in a human patient. A still further aspect of the inventionprovides a TIGIT antibody or antigen binding fragment as describedherein for use treating or preventing cancer in a human patient.

In another aspect the invention provides a method of selectivelydepleting Treg cells in a cancer patient, the method comprisingadministering an anti-TIGIT antibody or antigen-binding fragment thereofto the patient. In certain embodiments, the anti-TIGIT antibody binds atan epitope on human TIGIT comprising residues Q56, N58, E60, 168 L73,H76, and I109, preferably consisting of residues Q56, N58, E60, 168 L73,H76, and I109. In certain embodiments, the anti-TIGIT antibody is ananti-TIGIT antibody provided herein.

In certain embodiments, the anti-TIGIT antibody comprises a combinationof HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein: HCDR1 comprisesor consists SEQ ID NO: 16 (YTFTSYYMH), HCDR2 comprises or consists SEQID NO: 17 (VIGPSGASTSYAQKFQG), HCDR3 comprises or consists SEQ ID NO: 18(ARDHSDYWSGIMEV), LCDR1 comprises or consists SEQ ID NO: 61(RASQSVRSSYLA), LCDR2 comprises or consists SEQ ID NO: 62 (GASSRAT), andLCDR3 comprises or consists SEQ ID NO: 63 (QQYFSPPWT).

In certain preferred embodiments, the patient to be treated has a cancerselected from: renal cancer (e.g., renal cell carcinoma), breast cancer,brain tumours, chronic or acute leukaemias including acute myeloidleukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia,chronic lymphocytic leukaemia, lymphomas (e.g., Hodgkin's andnon-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma,B-cell lymphoma (e.g. CLL), T-cell lymphoma (e.g. Sezary Syndrome)),nasopharyngeal carcinomas, melanoma (e.g., metastatic malignantmelanoma), prostate cancer, colon cancer, lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck (e.g. headand neck squamous cell carcinoma (HNSCC)), cutaneous carcinoma,cutaneous or intraocular malignant melanoma, uterine cancer, ovariancancer, rectal cancer, cancer of the anal region, stomach cancer,testicular cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, cancer of the oesophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,solid tumours of childhood, cancer of the bladder, cancer of the kidneyor ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), tumour angiogenesis, spinal axis tumour, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer,squamous cell cancer, mesothelioma. In certain embodiments, the cancerinhibited is lung cancer, bladder cancer, breast cancer, kidney cancer(for example kidney carcinoma), head and neck cancer (e.g. HNSCC), orcolon cancer (for example colon adenocarcinoma). In certain embodiments,the cancer is colon cancer (for example colon adenocarcinoma) or lungcancer. In certain embodiments, the cancer is a blood cancer. In certainsuch embodiments, the cancer is lymphoma. In certain embodiments thecancer is T cell lymphoma or B cell lymphoma.

In certain embodiments, the method of treating cancer further comprisesadministration of an additional therapeutic agent, for example achemotherapeutic agent.

As demonstrated herein, the antibodies of the invention or antigenbinding fragments thereof are particularly effective when administeredin combination with immune checkpoint inhibitors—specifically anti-ICOSantagonist antibodies or anti-PD-1 antibodies (that is, antagonistantibodies specific for human immunoregulatory molecule PD-1).Administration of anti-TIGIT antibodies in combination with an anti-ICOSor anti-PD-1 antibody results in a synergistic reduction in tumourgrowth compared to either antibody alone. Similar effects are expectedto be observed using a combination of an anti-TIGIT antibody accordingto the invention and an anti-PD-L1 antibody.

It is further demonstrated herein that antibodies of the invention orantigen binding fragments thereof are particularly effective whenadministered in combination with an agonist antibody specific to animmune checkpoint co-stimulatory molecule—specifically anti-4-1BB,anti-OX40 or anti-GITR agonist antibodies. Administration of anti-TIGITantibodies in combination with an anti-4-1BB, anti-OX40 or anti-GITRagonist antibody results in a synergistic reduction in tumour growthcompared to either antibody alone.

Therefore, also provided herein is a method of treating cancer in asubject comprising administering to the subject an effective amount ofan anti-TIGIT antibody or antigen binding fragment thereof according tothe invention and also administering an effective amount of an anti-PD-1antibody, an anti-PD-L1 antibody, an anti-41BB antibody, an anti-OX40antibody, and anti GITR antibody, or an anti-ICOS antibody.

In addition, the data provided herein demonstrating that anti-TIGITantibodies can increase the activity of γδ cells as well as conventionalT cells indicates that anti-TIGIT antibodies can be used to treatconditions other than cancer. In particular, γδ T cells are known to beimportant in the response to infection, for example bacterial, fungal orviral infection. As shown in Example 29, when contacted with ananti-TIGIT antibody, γδ T cells from CMV seropositive subjects exhibitmarkedly increased activation, characterised by an increase in IFNgsection. The ability to promote activation of γδ T cells in CMV patientsin this manner indicates that administration of an anti-TIGIT antibodywill promote the antiviral activity of the γδ T cells.

Accordingly, provided herein is a method of treating viral infection ina subject comprising administering an effective amount of an anti-TIGITantibody or antigen-binding fragment thereof. Also provided is a methodof treating viral infection in a subject comprising administering aneffective amount of an anti-TIGIT antibody or antigen-binding fragmentor a pharmaceutical composition provided herein to the subject, therebytreating the viral infection. In preferred embodiments, the viralinfection is CMV infection.

In certain embodiments, the method further comprises administration ofone or more additional therapeutic agents. In certain embodiments, theone or more therapeutic agents are selected from: an anti-PD1 antibody,an anti-PD-L1 antibody, an anti-41BB antibody, an anti-OX40 antibody, ananti GITR antibody, and an anti-ICOS antibody.

As demonstrated in the Examples, the anti-TIGIT antibodies disclosedherein are effective at promoting T cell activity, especiallypro-inflammatory T cell activity. T ell activity can be measured bymethods familiar to those of skill in the art, for example by measuringIFNg production as described in the Examples.

Accordingly, also provided herein is a method of promoting T cellactivity comprising contacting a population of T cells with an antibodyor antigen binding fragment as described herein.

In certain embodiments, the method of promoting T cell activity isperformed in vitro. In certain embodiments, the method of promoting Tcell activity is performed in vivo in a human subject. In certain suchembodiments, the human subject has cancer. In certain embodiments, thehuman subject has a viral infection, for example CMV infection.

In certain embodiments, the method promotes conventional αβ T cellactivity. In certain embodiments, the method promotes CD4 T cellactivity. In certain embodiments, the method promotes CD8 T cellactivity. In certain embodiments, the method promotes γδ (gamma-delta) Tcell activity.

It is further demonstrated in the Examples that the anti-TIGITantibodies disclosed herein will be especially effective at promoting Tcell activity when used in combination with an anti-PD1 antibody, ananti-PD-L1 antibody, an anti-41BB antibody, an anti-OX40 antibody, ananti GITR antibody, or an anti-ICOS antibody. Significantly, thecombination provides a synergistic (i.e. greater than additive) increasein T cell activity.

Accordingly, in certain embodiments, the method of promoting T cellactivity further comprises contacting the population of T cells with oneor more of: an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-41BBantibody, an anti-OX40 antibody, an anti GITR antibody, and an anti-ICOSantibody.

Variants and equivalents of the embodiments of the invention describedherein but not departing from the spirit and scope of the invention willbe familiar to the skilled person. The invention will be furtherunderstood with reference to the following non-limiting Examples.

EXAMPLES Example 1: Selection of TIGIT Antigen-Binding Proteins

TIGIT ABPs were selected from a synthetic library of human antibodiesexpressed and presented on the surface of yeast cells in IgG formatgenerally as described, e.g., in WO2009036379; WO2010105256;WO2012009568; and Xu et al., Protein Eng Des Sel., Vol. 26(10), pp.663-670 (2013)), and more specifically as provided below. The sequencesand characteristics of the ABPs isolated from the recombinant libraryare provided in FIGS. 1 to 6 .

Eight naïve human synthetic yeast libraries each of ˜109 diversity werepropagated as described previously (see, e.g.: Xu et al, 2013;WO2009036379; WO2010105256; and WO2012009568). For the first two roundsof selection, a magnetic bead sorting technique utilizing the MiltenyiMACS system was performed, as described (see, e.g., Siegel et al.,2004). Briefly, yeast cells (˜1010 cells/library) were incubated withbiotinylated TIGIT-Fc antigen (Creative Biomart) in FACS wash buffer(phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA)). Afterwashing once with 50 ml ice-cold wash buffer, the cell pellet wasresuspended in 40 mL wash buffer, and Streptavidin MicroBeads (500 μl)were added to the yeast and incubated for 15 min at 4° C. Next, theyeast were pelleted, resuspended in 5 mL wash buffer, and loaded onto aMiltenyi LS column. After the 5 mL was loaded, the column was washed 3times with 3 ml FACS wash buffer. The column was then removed from themagnetic field, and the yeast were eluted with 5 mL of growth media andthen grown overnight. The following rounds of sorting were performedusing flow cytometry. Approximately 1×108 yeast were pelleted, washedthree times with wash buffer, and incubated with biotinylated TIGIT-Fcfusion antigen (10 nM) under equilibrium conditions at room temperature.Yeast were then washed twice and stained with LC-FITC (diluted 1:100)and either SA-633 (diluted 1:500) or EA-PE (diluted 1:50) secondaryreagents for 15 min at 4° C. After washing twice with ice-cold washbuffer, the cell pellets were resuspended in 0.4 mL wash buffer andtransferred to strainer-capped sort tubes. Sorting was performed using aFACS ARIA sorter (BD Biosciences) and sort gates were assigned to selectfor specific binders relative to a background control. Subsequent roundsof selection were employed in order to reduce the number of non-specificbinders utilizing soluble membrane proteins from CHO cells (See, e.g.,WO2014179363 and Xu et al., Protein Eng Des Sel, Vol. 26(10), pp.663-670 (2013)), and identify binders with improved affinity to TIGITusing the TIGIT-Fc antigen. After the final round of sorting, yeast wereplated and individual colonies were picked for characterization and fornomination of clones for affinity maturation. 63 clones were screenedfor functional activity. From the screening, clones 26518, 26452, 26486,26521 and 26493 had the best functional activity and were selected forfurther optimization.

Example 2: Antibody Optimization

Optimization of naïve clones was carried out utilizing three maturationstrategies: light chain diversification; diversification of HCDR1 andHCDR2; and diversification of HCDR3 within the selected HCDR1 and HCDR2diversity pools.

Light chain diversification: Heavy chain variable regions were extractedfrom naïve outputs (described above) and transformed into a light chainlibrary with a diversity of 1×106. Selections were performed asdescribed above with one round of MACS sorting and three rounds of FACSsorting using 10 nM or 1 nM biotinylated TIGIT-HIS antigen (CreativeBiomart) for respective rounds.

HCDR1 and HCDR2 selection: The HCDR3s from clones selected from thelight chain diversification procedure were recombined into a premadelibrary with HCDR1 and HCDR2 variants of a diversity of 1×108 andselections were performed using monomeric HIS-TIGIT antigen. Affinitypressures were applied by using decreasing concentrations ofbiotinylated HIS-TIGIT antigen (100 to 1 nM) under equilibriumconditions at room temperature.

HCDR3/HCDR1/HCDR2 selections: Oligos were ordered from IDT whichcomprised the HCDR3 as well as a homologous flanking region on eitherside of the HCDR3. Amino acid positions in the HCDR3 were variegated viaNNK diversity at two positions per oligo across the entire HCHR3. TheHCDR3 oligos were double-stranded using primers which annealed to theflanking region of the HCDR3. The remaining FWR1 to FWR3 of the heavychain variable region was amplified from pools of antibodies withimproved affinity that were isolated from the HCDR1 and HCDR2diversities selected above. The library was then created by transformingthe double stranded HCDR3 oligo, the FWR1 to FWR3 pooled fragments, andthe heavy chain expression vector into yeast already containing thelight chain of the original naïve parent. Selections were performed asduring previous cycles using FACS sorting for four rounds. For each FACSround the libraries were assessed for PSR binding, speciescross-reactivity, and affinity pressure, and sorting was performed toobtain populations with the desired characteristics. Affinity pressuresfor these selections were performed as described above in the HCDR1 andHCDR2 selection.

Example 3: Antibody Production and Purification

A. Production in Yeast

In order to produce sufficient amounts of optimized and non-optimizedselected antibodies for further characterization, the yeast clones weregrown to saturation and then induced for 48 h at 30° C. with shaking.After induction, yeast cells were pelleted and the supernatants wereharvested for purification. IgGs were purified using a Protein A columnand eluted with acetic acid, pH 2.0. Fab fragments were generated bypapain digestion and purified in a two steps process over Protein A (GELifeSciences) and KappaSelect (GE Healthcare LifeSciences).

B. Production in Mammalian Cells

In order to produce sufficient amounts of optimized and non-optimizedselected antibodies for further characterization, DNA vector coding forspecific antibody clones were generated and transduced into HEK cells.Human codon optimized synthetic DNA fragments for antibody variabledomains were ordered at Geneart. Variable domain sequences wereseamlessly ligated into pUPE expression vectors containing the mouseIgKappa signal sequence and constant regions of the respective antibodyclass. Expression vectors were verified by restriction analysis and DNAsequencing. For transient transfection Endotoxin free DNA maxipreps(Sigma) were produced and heavy and light chain vectors wereco-transfected to HEK293EBNA1 cells, in Freestyle medium(ThermoFisherScientific), according to established protocols. Primatone(0.55% final volume) was added 24 hour post-transfection. Conditionedmedium was harvested 6 days post transfection. Antibodies were purifiedbatch wise by Mabselect sureLX (GE Healthcare) affinity chromatography.Bound antibodies were washed in 2 steps with PBS containing 1M NaCl andPBS. Antibodies were eluted with 20 mM Citrate 150 mM NaCl pH3 andneutralized to approximately pH7 with ⅙ volume of 1M K2HPO4/KH2PO4 pH8.

Next the antibodies were further purified by gel-filtration using aSuperdex200 column, equilibrated in PBS. Fractions were analysed byNuPAGE and antibody containing fractions were pooled. The final productswere sterilized over a 0.22 μM syringe filter. The product was analysedby NuPAGE and endotoxin levels were measured by LAL-assay.

Example 4: Affinity Determination for Binding of Anti-TIGIT Antibodiesto Recombinant Human TIGIT Protein

A. ForteBio KD Measurements

ForteBio affinity measurements of selected antibodies were performedgenerally as previously described (see, e.g., Estep et al., Mabs, Vol.5(2), pp. 270-278 (2013)). Briefly, ForteBio affinity measurements wereperformed by loading IgGs on-line onto AHQ sensors. Sensors wereequilibrated off-line in assay buffer for 30 min and then monitoredon-line for 60 seconds for baseline establishment. Sensors with loadedIgGs were exposed to 100 nM antigen (human TIGIT-Fc, human TIGIT-His orcyno TIGIT-Fc) for 5 minutes, afterwards they were transferred to assaybuffer for 5 min for off-rate measurement. Kinetics were analyzed usingthe 1:1 binding model. More than 90 antibodies were tested for affinityby ForteBio and Table 3 provides data for 15 selected anti-TIGITantibodies demonstrating strong binding to recombinant TIGIT protein.

B. MSD-SET K_(D) Measurements

Equilibrium affinity measurements of selected antibodies were performedgenerally as previously described (Estep et al., Mabs, Vol. 5(2), pp.270-278 (2013)). Briefly, solution equilibrium titrations (SET) wereperformed in PBS+0.1% IgG-Free BSA (PBSF) with antigen (TIGIT-Hismonomer) held constant at 10-100 pM and incubated with 3- to 5-foldserial dilutions of Fab or mAbs starting at 10 pM-10 nM. Antibodies (20nM in PBS) were coated onto standard bind MSD-ECL plates overnight at 4°C. or at room temperature for 30 min. Plates were then blocked by BSAfor 30 min with shaking at 700 rpm, followed by three washes with washbuffer (PBSF+0.05% Tween 20). SET samples were applied and incubated onthe plates for 150s with shaking at 700 rpm followed by one wash.Antigen captured on a plate was detected with 250 ng/mL sulfotag-labeledstreptavidin in PBSF by incubation on the plate for 3 min. The plateswere washed three times with wash buffer and then read on the MSD SectorImager 2400 instrument using 1× Read Buffer T with surfactant. Thepercent free antigen was plotted as a function of titrated antibody inPrism and fit to a quadratic equation to extract the KD. To improvethroughput, liquid handling robots were used throughout MSD-SETexperiments, including SET sample preparation. Selected antibodies weretested for affinity by MSD and Table 4 provides data for 7 anti-TIGITclones demonstrating strong binding to recombinant TIGIT protein.

TABLE 4 MSD analysis of affinity for selected anti-TIGIT antibodiesClone MSD Affinity Monovalant KD (M) Human TIGIT-His 29489 1.1E−10 294947.0E−11 29499 1.9E−11 29513 2.5E−11 29520 2.1E−10 29523 1.7E−09 295276.4E−10

C. Biacore Measurement

Biosensor analysis was conducted at 25° C. in a HBS-EP buffer system (10mM HEPES pH 7.3, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20) using aBiacore 8K optical biosensor docked with a CM5 sensor chip (GEHealthcare, Marlboro, Mass.). The sample hotel was maintained at 8° C.Goat anti-human IgG capture antibody (Fcγ fragment specific, JacksonImmunoResearch Laboratories, Inc., West Grove, Pa.; 109-005-098) wasimmobilized (11700+/−200 RU) to both flow cells of the sensor chip usingstandard amine coupling chemistry. This surface type provided a formatfor reproducibly capturing fresh analysis antibody after eachregeneration step. Flow cell 2 was used to analyze captured antibody(60-90 RU) while flow cell 1 was used as a reference flow cell. Antigenconcentrations ranging from 30 to 0.123 nM (3-fold dilutions) wereprepared in running buffer. Each of the antigen sample concentrationswere run as a single replicate. Two blank (buffer) injections also wererun and used to assess and subtract system artefacts. The association(300 s) and dissociation (600 s) phases for all antigen concentrationswere performed at a flow rate of 30 μl/min. The surface was regeneratedwith three sequential injections (15 s, 15 s and 60 s) of 10 mM glycine,pH 1.5 at a flow rate of 30 μl/min. The data was aligned, doublereferenced, and fit to a 1:1 binding model using Biacore 8K EvaluationSoftware, version 1.0. Selected antibodies were tested for affinity byBiacore and Table 5 provides data for 5 anti-TIGIT clones demonstratingstrong binding to recombinant TIGIT protein.

TABLE 5 Biacore analysis of affinity for selected anti-TIGIT antibodiesBiacore: Monovalent KD (M) (IgG on CM5 chip, Human TIGIT-HIS in solutionClone (Starting concentration 25 nM, 3x dilution) 29489 2.48E−10 312822.94E−10 29494 2.70E−10 29520 7.16E−10 29527 1.20E−09 31288 1.92E−10

Example 5: Competition Assay Between Anti-TIGIT Antagonistic Antibodiesand TIGIT Natural Ligands

A. Octet Red384 Epitope Binning/Ligand Blocking

Epitope binning/ligand blocking of selected antibodies was performedusing a standard sandwich format cross-blocking assay. Controlanti-target IgG was loaded onto AHQ sensors and unoccupied Fc-bindingsites on the sensor were blocked with an irrelevant human IgG1 antibody.The sensors were then exposed to 100 nM target antigen (hTIGIT, CreativeBiomart) followed by a second anti-target antibody or ligand (anti-TIGITantibody and CD155 or CD113 or CD112). Data was processed usingForteBio's Data Analysis Software 7.0. Additional binding by the secondantibody or ligand after antigen association indicates an unoccupiedepitope (non-competitor), while no binding indicates epitope blocking(competitor or ligand blocking). Parental antibodies (beforeoptimization) were tested for competition with natural ligands and Table6 summarizes the data obtained for competition against CD155, CD112 andCD113. Parental clone 26432 was found not to compete with CD155 forTIGIT binding. All other selected anti-TIGIT antibodies compete withnatural ligand for binding to recombinant human TIGIT protein.

TABLE 6 Binning analysis against TIGIT natural ligands for non-optimizedanti-TIGIT antibodies CD155 CD112 CD113 Clone competition competitioncompetition 26518 Yes Yes Yes 26452 Yes Yes Yes 26486 Yes Yes Yes 26521Yes Yes Yes 26493 Yes Yes Yes 26432 No

B. Competition of Anti-TIGIT Antagonistic Antibodies with CD155 onJurkat-hTIGIT

Jurkat cells overexpressing human TIGIT (Jurkat-hTIGIT) were collectedand distributed at 10⁵ cells/well and incubated with anti-human TIGITantibodies at the following concentrations: 166.6; 53.24; 17.01; 5.43;1.73; 0.55; 0.17; 0.05; 0.01; 5.78×10⁻³; 1.85×10⁻³; 5.9×10⁻³ nM incomplete medium during 45 min at 37° C. Excess of antibody was washed,and then the cells were incubated with CD155-His at 5 μg/ml (CreativeBiomart, PVR-3141H) for 45 min at 37° C. Then, bound CD155-His wasdetected using anti-His tag-PE (Biolegend, 362603, at 2 μl per test),incubated for 30 min at 4° C. Cells were analysed by FACS using BDLSRFortessa and the half concentration (IC50) that prevents CD155binding was calculated on the basis of the geometric mean fluorescence.

Jurkat cells overexpressing human TIGIT (Jurkat-hTIGIT) were collectedand distributed at 105 cells/well and incubated with anti-human TIGITantibodies at the following concentrations: 166.6; 53.24; 17.01; 5.43;1.73; 0.55; 0.17; 0.05; 0.01; 5.78×10-3; 1.85×10-3; 5.9×10−3 nM incomplete medium during 45 min at 37° C. Excess of antibody was washed,and then the cells were incubated with CD155-His at 5 μg/ml (CreativeBiomart, PVR-3141H) for 45 min at 37° C. Then, bound CD155-His wasdetected using anti-His tag-PE (Biolegend, 362603, at 2 μl per test),incubated for 30 min at 4° C. Cells were analysed by FACS using BDLSRFortessa and the half concentration (IC50) that prevents CD155binding was calculated on the basis of the geometric mean fluorescence.

The results were as follows: 0.101 nM for clone 29489; 0.07 nM for clone29494; 0.102 nM for clone 29520 and 0.078 nM for clone 29527, for theresults illustrated in FIG. 7 . The values of other tested antibodiesare summarized in the Table 7 below. Overall, the results demonstrate astrong competition by the tested antagonistic anti-TIGIT antibodies withCD155 for binding to membrane expressed TIGIT.

TABLE 7 IC₅₀ data for CD155 competition on human TIGIT IC50 of CD155competition Clone for TIGIT binding (in nM) 29489 0.101 29494 0.07029499 0.103 29513 0.094 29520 0.102 29523 0.079 29527 0.078

Example 6: Characterization of Hydrophobic Interaction Chromatography(MAbs. 2015 May-June; 7(3): 553-561.)

Anti-TIGIT IgG1 antibody samples were buffer exchanged into 1 M ammoniumsulfate and 0.1 M sodium phosphate at pH 6.5 using a Zeba 40 kDa 0.5 mLspin column (Thermo Pierce, cat #87766). A salt gradient was establishedon a Dionex ProPac HIC-10 column from 1.8 M ammonium sulfate, 0.1 Msodium phosphate at pH 6.5 to the same condition without ammoniumsulfate. The gradient ran for 17 min at a flow rate of 0.75 ml/min. Anacetonitrile wash step was added at the end of the run to remove anyremaining protein and the column was re-equilibrated over 7 columnvolumes before the next injection cycle. Peak retention times weremonitored at A280 absorbance and concentrations of ammonium sulfate atelution were calculated based on gradient and flow rate. Table 8summarizes the results obtained for 15 selected anti-TIGIT antibodies.

TABLE 8 Analysis of Hydrophobic Interaction Chromatography for selectedanti-TIGIT antibodies Hydrophobic Interaction Chromatography Clone (HIC)Retention Time (min) 26518 10.4 29478 12.7 26452 9.3 29487 9.9 2948910.6 26486 11.0 29494 9.7 29499 9.1 26521 12.4 29513 12.5 26493 8.829520 9.6 29523 8.7 29527 8.6 26432 11.1 32919 9.0 32931 9.3 32959 12.0

Example 7: Characterization of PSR Preparation Polyspecificity Reagent

A. Preparation of Polyspecificity Reagent:

Polyspecificity reagent (PSR) was prepared according to Xu et. al, mAbs2013. In brief, 2.5 liters CHO-S cells were used as starting material.The cells were pelleted at 2,400×g for 5 min in 500 mL centrifugebottles filled to 400 mL. Cell pellets were combined and thenresuspended in 25 ml Buffer B and pelleted at 2,400×g for 3 min. Thebuffer was decanted and the wash repeated one time. Cell pellets wereresuspended in 3× the pellet volume of Buffer B containing 1× proteaseinhibitors (Roche, cOmplete, EDTA-free) using a polytron homogenizerwith the cells maintained on ice. The homogenate was then centrifuged at2,400×g for 5 min and the supernatant retained and pelleted oneadditional time (2,400×g/5 min) to ensure the removal of unbroken cells,cell debris and nuclei; the resultant supernatant is the total proteinpreparation. The supernatant was then transferred into two Nalgene OakRidge 45 mL centrifuge tubes and pelleted at 40,000×g for 40 min at 4°C. The supernatants containing the Separated Cytosolic Proteins (SCPs)were then transferred into clean Oak Ridge tubes, and centrifuged at40,000×g one more time. In parallel, the pellets containing the membranefraction (EMF) were retained and centrifuged at 40,000 for 20 min toremove residual supernatant. The EMF pellets were then rinsed withBuffer B. 8 mL Buffer B was then added to the membrane pellets todislodge the pellets and transfer into a Dounce Homogenizer. After thepellets were homogenized, they were transferred to a 50 mL conical tubeand represented the final EMF preparation.

One billion mammalian cells (e.g. CHO, HEK293, Sf9) at ˜106-107 cells/mLwere transferred from tissue culture environment into 4×250 mL conicaltubes and pelleted at 550×g for 3 min. All subsequent steps wereperformed at 4° C. or on ice with ice-cold buffers. Cells were washedwith 100 mL of PBSF (1×PBS+1 mg/mL BSA) and combined into one conicaltube. After removing the supernatant, the cell pellet was thenre-suspended in 30 mL Buffer B (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl2, 5mM KCl, 5 mM MgCl2, 10% Glycerol, pH 7.2) and pelleted at 550×g for 3min. Buffer B supernatant was decanted and cells re-suspended in 3×pellet volume of Buffer B plus 2.5× protease inhibitor (Roche, cOmplete,EDTA-free). Protease inhibitors in Buffer B were included from here onforward. Cells were homogenized four times for 30 sec pulses (Polytonhomogenizer, PT1200E) and the membrane fraction was pelleted at 40,000×gfor 1 hour at 4 C. The pellet is rinsed with 1 mL Buffer B; thesupernatant is retained and represents the s. The pellet is transferredinto a Dounce homogenizer with 3 mL of Buffer B and re-suspended bymoving the pestle slowly up and down for 30-35 strokes. The enrichedmembrane fraction (EMF) is moved into a new collection tube, rinsing thepestle to collect all potential protein. Determine the proteinconcentration of the purified EMF using the Dc-protein assay kit(BioRad). To solubilize the EMF, transfer into Solubilization Buffer (50mM HEPES, 0.15 M NaCl, 2 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 1%n-Dodecyl-b-D-Maltopyranoside (DDM), 1× protease inhibitor, pH 7.2) to afinal concentration of 1 mg/mL. Rotate the mixture overnight at 4° C.rotating followed by centrifugation in a 50 mL Oak Ridge tube (FisherScientific, 050529-ID) at 40,000×g for 1 hour. The supernatant, whichrepresents the soluble membrane proteins (SMPs), was collected and theprotein yield quantified as described above.

For biotinylation, prepare the NHS-LC-Biotin stock solution according tomanufacturer's protocol (Pierce, Thermo Fisher). In brief, 20 μl ofbiotin reagent is added for every 1 mg of EMF sample and incubated at 4°C. for 3 hours with gentle agitation. Adjust the volume to 25 mL withBuffer B and transfer to an Oak Ridge centrifuge tube. Pellet thebiotinylated EMF (b-EMF) at 40,000×g for 1 hour, and rinse two timeswith 3 mL of Buffer C (Buffer B minus the glycerol) without disturbingthe pellet. Remove the residual solution. The pellet was re-suspendedwith a Dounce homogenizer in 3 mL of Buffer C as described previously.The re-suspended pellet now represents biotinylated EMF (b-EMF) and issolubilized as described above to prepare b-SMPs.

B. PSR Binding Analyses

PSR analyses were carried out generally as described in WO2014/179363.Briefly, to characterize the PSR profile of monoclonal antibodiespresented on yeast, two million IgG-presenting yeast were transferredinto a 96-well assay plate and pellet at 3000×g for 3 min to removesupernatant. Re-suspend the pellet in 50 μl of freshly prepared 1:10dilution of stock b-PSRs and incubate on ice for 20 minutes. Wash thecells twice with 200 μl of cold PBSF and pellet re-suspended in 50 μl ofsecondary labeling mix (Extravidin-R-PE, anti-human LC-FITC, andpropidium iodide). Incubate the mix on ice for 20 minutes followed bytwo washes with 200 μl ice-cold PBSF. Re-suspend the cells in 100 μl ofice-cold PBSF and run the plate on a FACS Canto (BD Biosciences) usingHTS sample injector. Flow cytometry data was analyzed for meanfluorescence intensity in the R-PE channel and normalized to propercontrols in order to assess non-specific binding. Table 9 summarizes theresults of Poly-specificity Reagent binding obtained for 15 selectedanti-TIGIT antibodies which confirm low score for most of the clones.

TABLE 9 Analysis of Polyspecificity Reagent Clone PolyspecificityReagent (PSR) Score (0-1) 26518 0.00 29478 0.01 26452 0.00 29487 0.0129489 0.01 26486 0.00 29494 0.00 29499 0.10 26521 0.00 29513 0.01 264930.00 29520 0.32 29523 0.12 29527 0.12 26432 0.00 31288 0.00 32919 0.0032931 0.00 32959 0.1

Example 8: Characterization of TIGIT Expression on Immune Populationsfrom Healthy Human PBMC

A. TIGIT Expression Profile on T Cell Subsets

Flow cytometry analyses were performed to assess the expression of TIGITon immune cell subsets in PBMC freshly isolated from healthyindividuals. Conjugated antibodies were purchased fromEbioscience/Thermo Fisher Scientific, BioLegend or BD Biosciences. Cellswere stained per manufacturer's instruction using filtered FACS buffer(PBS+2 mM EDTA+0.1% BSA) and Brilliant Stain buffer (BD #563794). Cellswere blocked with appropriate Human FcBlock (BD #564220) prior tostaining and were fixed using IC fixation buffer (eBioscience#00-8222-49) prior acquisition. Acquisition was performed on a FACSFortessa (BD Biosciences) and analyzed with FlowJo software (FlowJo,LLC). Viable cells were gated on Forward and Side scatter. VariousImmune cells subsets were gated as followed: CD19+(B cells), CD3− CD19−CD14+ (Monocytes), CD3+ TCRab− (TCRgd T cells), CD3+ TCRab+ (TCRab Tcells), CD3− CD19− CD14− HLA-DR− CD56low/high (NK cells), CD3− CD19−CD14− HLA-DR+ (Dendritic cells), CD3+ TCRab+ CD4+ CD127low CD25+(regulatory T cells), CD3+ TCRab+ CD4+ or CD8+ CD45RO− CCR7+ (CD4 or CD8naïve T cells), CD3+ TCRab+ CD4+ or CD8+ CD45RO+ (memory T cells) andCD45RO−CD62L− (effector T cells),

As shown in FIGS. 8A and 8B, TIGIT is preferentially expressed on NKcells, regulatory T cells and CD8 memory T cells. It is present to alesser extent on other T cells subsets with a low proportion of naïve Tcells showing TIGIT expression. In addition, TIGIT is not expressed onmonocytes, dendritic cells and B cells (FIG. 8B). This set of data is inagreement with published data (Yu et al. NI 2008 and Wang et al. EJI2015).

Example 9: Cellular Binding of Anti-TIGIT Antagonistic Antibodies

A. Binding of Anti-TIGIT Antibodies to Jurkat-hTIGIT and Jurkat-mTIGIT

The affinity of human anti-TIGIT antibodies has been measured usingJurkat E6.1 cells transduced with human-TIGIT (Jurkat hTIGIT) or mouseTIGIT (Jurkat-mTIGIT). To analyse the affinity of the selectedantibodies for hTIGIT or mTIGIT, 105 cells were distributed per well andincubated with anti-TIGIT antibody at a single dose of 100 nM (Table 3)or with decreasing concentration (166.6; 53.24; 17.01; 5.43; 1.73; 0.55;0.17; 0.05; 0.01; 5.78×10⁻³; 1.85×10⁻³; 5.9×10⁻³ nM) of selectedantibodies (FIG. 9 ). Antibodies were incubated with the cells for 20min at 4° C. in FACS buffer. After washing, cells were incubated withanti-human Ig (Fc gamma specific)-PE (eBioscience, 12-4998-82, at 2.5μg/ml) for 20 min on ice and washed twice. Geometric mean fluorescenceintensity was analysed using LSR BD Fortessa. Cell binding was recordedas the median florescence intensity of PE on the transfected linecompared to the un-transfected line for each antibody (Table 3). Forcalculation of EC50 binding, the half-maximal concentration of binding(EC50) to hTIGIT-Jurkat was calculated using a four-variable curve-fitequation in Prism, and the obtained values were the following ones:0.082 nM for clone 29489; 0.07 nM for clone 29494; 0.119 nM for clone29520 and 0.05 nM for clone 29527 for the data illustrated in FIG. 9 .The results demonstrate a strong binding to membrane expressed humanTIGIT for the tested anti-TIGIT antibodies.

B. Binding of Anti-TIGIT Antagonistic Antibodies to Human Primary TCells

Isolated human PBMCs from healthy volunteers were analysed for bindingby antagonistic anti-TIGIT antibodies. Cells were distributed at 5×105cells per well. Cells were incubated with anti-CD16 (Clone 3G8,BioLegend 302002), CD32 (Clone FLI8.26, BD Bioscience 557333) and CD64(BD Bioscience 555525) at room temperature for 10 min, and the indicatedanti-human TIGIT antibodies were directly added at a final concentrationof: 12.65; 4; 1.26; 0.40; 0.126; 0.040; 0.12 and 4×10⁻³ nM in FACSbuffer and incubated for 20 min at 4° C. After washing, cells wereincubated with anti-human Ig (Fc gamma specific)-PE (eBioscience,12-4998-82, at 2.5 μg/ml) for 20 min at 4° C. Then, cells were washedand incubated with the following antibodies and LVD mix for results ofFIGS. 10A and 10B: anti-CD4-PercP-Cy5.5 (clone A161A1, BioLegend357414); anti-CD8-BV510 (clone SK1, BD Bioscience 563919) and LVD efluor520 (eBioscience 65-0867-14). For FIG. 10C, cells were washed andincubated with the following antibodies and LVD mix: LVD efluor 520(eBioscience 65-0867-14), anti-TCRab-PercP-Cy5.5 (Clone IP26, Biolegend306723), anti-CD4-BV510 (Clone SK3, BD Horizon 562970), anti-CD8-APC-Cy7(Clone SK1, Biolegend 344714), anti-CD25-BV605 (Clone 2A3, Biolegend562660), anti-CD127-APC (A019D5, Biolegend 351316), anti-CCR7-BV421(Clone G043H7, Biolegend 353207) and anti-CD45RO-PE-Cy7 (Clone UCHL1,Biolegend 304229).

The EC50 value for binding to CD8+ human primary T cells was calculatedusing the % of positive TIGIT stained cells on gated LVD-CD8+ T cells(FIGS. 10A and 10B). The EC50 value for binding to human memory CD8+ orTreg primary T cells was calculated using the % of positive TIGITstained cells on gated LVD-TCRab+CD45RO+CD8+ T cells (for memory CD8+ Tcells) or on gated LVD-TCRab+CD127loCD25hiCD4+ T cells (for Tregs) andare illustrated in FIG. 10C.

As shown in FIG. 10A, the EC50 value for binding to total human CD8+ Tcells are 0.123 nM for clone 29489; 0.181 nM, for clone 29520 and 0.253nM for clone 29527. Direct comparison between 29489 and 31282 (the 29489mutant with a M to T mutation on residue 116) was performed, and theEC50 value was 0.057 nM and 0.086 nM respectively, demonstrating strongand similar binding efficacy to human primary CD8+ T cells for the 2clones (FIG. 10B). The EC50 values obtained for binding to memory CD8+ Tcells and Treg were 0.039 nM and 0.03 nM respectively, demonstrating astrong and similar affinity for both populations (FIG. 10C).

C. Binding of Anti-TIGIT Antagonistic Antibodies to Cynomolgus Primary TCells

Isolated PBMCs from Macaca fascicularis were obtained from BioPRIM.Cells were thawed and stimulated using the T cell activation/expansionkit for non-human primate (Miltenyi Biotec) at 1:2 (bead:cell ratio)following the manufacturer's specifications. The next day, cells werecollected, counted and distributed at 5×10⁴ cells per well. Cells wereincubated with anti-CD16 (Clone 3G8, BioLegend 302002), CD32 (CloneFLI8.26, BD Bioscience 557333) and CD64 (BD Bioscience 555525) at roomtemperature for 10 min, and selected anti-human TIGIT antibodies weredirectly added at a final concentration of: 12.65; 4; 1.26; 0.40; 0.126;0.040; 0.12 and 4×10⁻³ nM in FACS buffer and incubated for 20 min at 4°C. After washing, cells were incubated with anti-human Ig (Fc gammaspecific)-PE (eBioscience, 12-4998-82, at 2.5 μg/ml) for 20 min at 4° C.Then, cells were washed and incubated with the following antibodies andLVD mix for data illustrated in FIGS. 11A and 11B: anti-CD4-PercP-Cy5.5(clone A161A1, BioLegend 357414); anti-CD8-BV510 (clone SK1, BDBioscience 563919), CD69-APC-Cy7 (Clone FN50, BioLegend, 310914) and LVDefluor 520 (eBioscience 65-0867-14). Stained cells were analysed by FACSusing BD LSR Fortessa. The EC50 value of binding was calculated usingthe % of positive TIGIT stained cells gated on LVD-CD69+CD8+ T cells. Asshown in FIG. 11 , the EC50 values for binding to cynomolgus CD8+ Tcells were 0.487 nM for clone 29489, 1.73 nM for clone 29520 and 0.378nM for clone 29527. Clones 29489 and 31282 (the 29489 mutant with a M toT mutation on residue 116) were compared as well, and the EC50 valueswere 0.25 nM and 0.26 nM respectively for the example shown in FIG. 11B,demonstrating a similar and strong affinity for cynomolgus primary CD8+T cells for the 2 clones.

Example 10: In Vitro Functional Characterization of AntagonisticAnti-TIGIT Activity

A. TIGIT Bioassay on CHO-TCR-CD155 and Jurkat-hTIGIT Co-Culture

To characterize the functional consequence of blocking human TIGITreceptor, we co-cultured Jurkat cells, that express hTIGIT and aluciferase reporter activated upon TCR engagement (Thaw-and-Use TIGITEffector cells from Promega), with CHO-K1 cell line engineered toexpress human CD155 and TCR activator (Thaw-and-Use CD155 aAPC/CHO-K1from Promega). The activation of TIGIT-overexpressing Jurkat cells canbe induced by contact with CD155-expressing CHO-K1 cells upon TCRengagement on Jurkat cells and can be increased in presence ofantagonistic anti-TIGIT antibody. To compare the potency of thedifferent antibodies to increase Jurkat cell activation, the experimentwas conducted in presence of increasing antibody concentrations and theEC50 values were calculated.

Thaw-and-Use CD155 aAPC/CHO-K1 (Promega, CS198811) cells were seededaccording to manufacturer's recommendations and incubated at 37° C., 5%CO2 incubator 0/N. The day after, Thaw-and-Use TIGIT Effector cells(Promega, CS198811) were added according to manufacturer'srecommendations to the CD155 aAPC/CHO-K1 cell plates containing freshfull medium with anti-TIGIT antibody at 133 nM (FIG. 12A) or increasingconcentrations (0.22; 0.54; 1.36; 3.41; 8.53; 21.3; 53.3; 133.33; and333 nM) of anti-TIGIT antibody (FIG. 12B) and incubated at 37° C., 5%CO2 during 6 hours.

After the 6 hours of incubation, activation of TIGIT Effector cell wasassessed by measuring the luciferase activity by using Bio-Glo™Luciferase Assay System (Promega, G7941).

FIG. 12A shows the effect of the addition of the selected clones onLuciferase signal as compared to isotype control. The data demonstratesthe antagonistic activity of those antibodies that resulted in astronger activation of Jurkat-hTIGIT cells. Table 10 summarizes the foldchange induction in luciferase expression obtained for the differentanti-TIGIT antibodies over the isotype control clone (03847).

TABLE 10 Fold change induction over isotype control Clone name Inductionover Isotype control (fold change) 26518 2.89 29478 3.57 26452 2.9 294873.22 29489 4.08 26486 1.9 29494 3.42 29499 3.68 26521 2.66 29513 3.2626493 0.96 29520 2.52 29523 2.4 29527 2.96 03847 1

As shown in FIG. 12B, Jurkat-hTIGIT cell activation was assessed withanti-TIGIT antibody between 0.22 nM and 333 nM and gave an EC50 value of3.0 nM for clone 29489; 4.4 nM for clone 29494; 2.3 nM for clone 29520and 32 nM for clone 29527; 2.7 nM for clone 32919 and 3.2 nM for clone32931 demonstrating a strong functional activity consecutive to blockingTIGIT inhibitory signalling. Clones 29489 and 31282 (the 29489 mutantwith a M to T mutation on residue 116) were compared as well, and theEC50 values were respectively of 4.3 nM and 8.1 nM for the example shownin FIG. 12C, demonstrating a similar functional activity for the 2clones.

B. Human Primary CD8⁺ T Cell-Based Functional Assay

To characterize the functional consequence of blocking human TIGITreceptor, we co-cultured human primary CD8+ T cells from PBMC of healthyhuman donors with CHO-K1 cell line engineered to express human CD155 andto activate human T cells. We observed that the release of IFNg by CD8+T cells in the presence of engineered CD155-expressing CHO-K1 cellscould be increased by blocking hTIGIT with anti-TIGIT antagonisticantibodies. To compare the potency of these antibodies to increase IFNgrelease, the experiment was conducted in presence of increasing antibodyconcentrations and the EC50 values were calculated.

Thaw-and-Use CD155 aAPC/CHO-K1 (Promega, CS198811) cells were seeded inU-bottom 96-well plates according to manufacturer's recommendations andincubated at 37° C., 5% CO2 incubator 0/N. The next day, CD8+ T cellswere purified according to manufacturer's recommendations by usingnegative selection kit (Stemcell Technologies, 17953) from frozen humanperipheral blood mononuclear cells isolated from total blood of healthydonors (Immunehealth). Purified CD8 T cells were then incubated withincreasing concentrations (0.11 nM, 0.33 nM, 1.06 nM, 3.3 nM, 10.6 nM,33.3 nM, 105.5 nM and 333 nM) of antibodies (100,000 CD8 T cells/100W offull medium containing antibody) during 1 hour. After that, theantibody-CD8 mix was added to the CD155 aAPC/CHO-K1 cell platescontaining 50 μl of fresh full medium and incubated at 37° C., 5% CO2during 5 days. Finally, IFNg concentrations were assessed in cellsupernatant using an ELISA assay (Affymetrix eBioscience, 88-7316-86)that was run according to manufacturer's recommendations.

As shown in FIG. 13A, all the anti-TIGIT antibodies increased IFNgsecretion over isotype control. The highest increase was observed withclone 29489 (6.4 fold) followed by 29494 (5.8 fold), 29520 (5.4 fold),29499 (5.2 fold), 29527 (4.5 fold) and 29513 (3.2 fold).

Dose range study (between 0.22 nM and 333 nM of anti-TIGIT antibody) wasalso conducted to evaluate the EC50 value for increase in IFNg secretionby human primary CD8 T cells. As shown in FIG. 13B, anti-TIGIT antibody29489 showed the best activity with an EC50 of 3.5 nM followed by clone29527 EC50=5.1 nM), clone 29494 (EC50=6.1 nM) and clone 29520 (EC50=11.1nM). Finally, clone 29489 and its mutant 31282 were tested in paralleland demonstrated a similar activity with a respective EC50 value of 0.49nM and 0.50 nM (FIG. 13C). Altogether these data demonstrate a strongfunctional activity of antagonistic anti-TIGIT antibodies to block TIGITinhibitory signal in CD8+ human T cells and to increase effectorfunctions, as characterized by a strong increase in IFNg production.

C. Human TIL Functional Assay

To characterize the functional consequence of blocking human TIGITreceptor on Tumour Infiltrating Lymphocytes (TILS) from cancer patients,we co-cultured human primary CD8+ T cells from TILs of ovarian ascitespatient with CHO-K1 cell line engineered to express human CD155 and toactivate human T cells. We observed that the release of IFNg by CD8+ Tcells in presence of engineered CD155-expressing CHO-K1 cells can beincreased by blocking hTIGIT with anti-TIGIT antagonistic antibodies.

Thaw-and-Use CD155 aAPC/CHO-K1 (Promega, CS198811) cells were seeded inU-bottom 96-well plates according to manufacturer's recommendations andincubated at 37° C., 5% CO2 incubator 0/N. The next day, CD8 T cellswere purified according to manufacturer's recommendations by usingnegative selection kit (Stemcell Technologies, 17953) from frozen humanTILs isolated from ovarian ascites (Immunehealth). Purified CD8+ T cellswere then incubated with anti-TIGIT antibody clone 26452, thenon-optimized parent of clones 29489 and 31282 (100,000 CD8+ Tcells/100W of full medium containing antibody) during 1 hour. Afterthat, the antibody-CD8 mix was added to the CD155 aAPC/CHO-K1 cellplates containing 50 μl of fresh full medium and incubated at 37° C., 5%CO2 during 5 days.

Finally, IFNg concentrations were assessed in cell supernatant using anELISA assay (Affymetrix eBioscience, 88-7316-86) that was run accordingto manufacturer's recommendations. As seen in FIG. 14 , IFNg secretionwas increased by almost 2 folds when anti-TIGIT antibody was added tothe co-culture. These data demonstrate a strong functional activity ofantagonistic anti-TIGIT antibodies to block TIGIT inhibitory signal inCD8+ human TILs and to increase effector functions of T cells in atumour setting.

Example 11: Characterization of Anti-TIGIT Antagonistic Antibody withFunctional Activity in Mouse

A. Mouse CD155 Competition Assay for Surrogate Anti-TIGIT AntagonisticAntibody

For this assay, Jurkat cells (clone E6-1, ATCC TIB-152) engineered tooverexpress mouse TIGIT (Jurkat-mTIGIT) were used. Anti-TIGIT antibody26493 was used as a surrogate as this antibody showed cross-reactivityfor mouse TIGIT as well as binding to human TIGIT. Cells werepre-incubated for 45 min at 37° C. with different concentrations ofanti-TIGIT antibody clone 26493 (0.03 to 10 μg/ml) in 25 μl of completemedium (RPMI+10% FBS). Cells were washed once and incubated with 4 μg/mlmouse CD155-His-Fc tag protein (Thermo Fisher, 50259M03H50) in 50 μl ofcomplete medium for 45 min in incubator. Cells were washed once, andstained with PE-anti-His antibody (Biolegend, 362603) during 30 min at4° C. The median fluorescence intensity (MFI) measured by FACS was usedas a measure of binding of CD155 to Jurkat-mTIGIT. FIG. 15A shows thedose-response curve of anti-TIGIT clone 26493 for CD155 competitionidentifying 2.3 nM as IC50 (upper dotted line represent signal fromisotype, bottom dotted line signal from cells without CD155). Theseresults demonstrate the functional efficacy of anti-TIGIT antibody tocompete with CD155 ligand for mouse TIGIT.

B. Mouse Functional In Vitro Assay: Antigen-Specific Cytotoxicity (OT-I)

To assess the antigen-specific cytotoxic activity of OT-I CD8+ T cellstowards OVA-pulsed target cells and the effect of anti-TIGIT antibody inthis assay, OT1 cells were isolated from the spleens ofC57BL/6-Tg(TcraTcrb)1100Mjb/Crl mice (Charles River) by mechanicaldissociation followed by negative selection for mouse T cells usingEasySep™ Mouse T Cell Isolation Kit (Stemcell, Catalog #19851). Asantigen-presenting cells, PanO2 cancer cells that naturally expressCD155, were treated with Mitomycin C (25 μg/ml) and subsequently pulsedwith OVA-peptide (S7951-1MG, Sigma Aldrich, 1 μg/ml, 1 h at 37° C.).CD8+ T cells and PanO2 were co-cultured for 3 days in the presence ofanti-TIGIT clone 26493 or isotype control at 133 nM. At day 3,supernatant was collected for detection of IFNg by ELISA (FIG. 15B) andT cells for the cytotoxicity assay (FIG. 15C). As target cells,OVA-pulsed PanO2 were used. Target cells and non-pulsed PanO2 cells(non-target internal control), 1×106 each, were labelled with CFSE(C1157, ThermoFisher) and CellTrace™ Far Red Cell Proliferation Kit(C34564, ThermoFisher) respectively, according to manufacturerinstructions. These cells were mixed (1:1 ratio) and plated at 2×104cells per well. The stimulated OT-1 CD8+ T cells were added at 1×105cells/well (effector cells) resulting in 10:1 effector to target ratioin the presence of anti-TIGIT clone 26493 or isotype control at 133 nM.After 24 hrs cells were washed with PBS and lifted by trypsinization.Cells were then stained with Live/dead fixable violet dead cell stainingkit (Molecular Probes, L34955). Cytotoxic killing of target cells wasthen measured by monitoring the change in the ratio of living targetcells to non-target cells by flow-cytometry.

FIG. 15B shows that anti-TIGIT antibody increases IFNg production byalmost 2 folds while FIG. 15C shows an increased cytotoxic activity ofmouse OT-I CD8+ T cells of around 60%. Altogether, these results confirmthe functional activity of anti-TIGIT antibody to increase mouse CD8+ Tcell effector function.

Example 12: Anti-Tumour Activity of Anti-TIGIT Antagonistic Antibody inMonotherapy and in Combination with Anti-PD1 Antibodies in Mouse Model

A. In Vivo Anti-Tumor Activity of Anti-TIGIT Antagonistic Antibody inMonotherapy

For this experiment, anti-TIGIT clone 26493 was produced in mammaliancells on a mouse IgG2a isotype. Female Balb/c mice of 8 weeks wereinoculated with 500.000 CT26 colon cancer cells (ATCC® CRL2638™)subcutaneously. On day 9 after inoculation, when tumor volumes were onaverage around 45 mm3, mice were randomized in treatment groups withequal tumor volume (n=8 per group). Mice were treated with 200 μg ofanti-TIGIT or with isotype control (mIgG2a, BioXcell) or with 200 μg ofanti-PD-1 (RMP1-14, BioXcell) and 200 μg of isotype control (mIgG2a,BioXcell) or with 200 μg of anti-PD-1 (RMP1-14, BioXcell) and differentconcentrations of anti-TIGIT (200 μg, 60 μg, 20 μg) by intraperitonealinjections on day 9, day 12 and day 15. Tumor growth was monitored andtumor volumes were measured with electronic calipers three times a weekfrom day 9 until day 36. Mice were sacrificed when tumor volume exceeded2000 mm3. Tumor growth curves were statistically analyzed by a linearmixed model. Differences between treatment groups were evaluated bytesting the interaction of time*treatment group. To test for asynergistic effect between anti-TIGIT and anti-PD-1, treatment groupswere recoded by a combination of two variables; anti-TIGIT (yes/no) andanti-PD-1 (yes/no). A synergistic effect, on top of the additive effectof each treatment (anti-TIGIT*time and anti-PD-1*time) was evaluated bytesting the interaction term anti-TIGIT*anti-PD-1*time.

FIG. 16A shows median tumor growth curves per group as well asindividual growth curves for mice treated with anti-TIGIT antibody inmonotherapy. Whereas in the control group, no mice had regression of thetumor, 2/8 mice treated with anti-TIGIT had a complete response. In theremaining mice, a clear tumor growth delay was present. In the controlgroup, no mice survived beyond 30 days, whereas in the treated group,7/8 mice survived beyond 30 days.

FIG. 16B shows median tumor growth curves per group as well asindividual growth curves for mice treated by anti-PD1 in monotherapy orin combination with anti-TIGIT. There was significant suppression oftumor growth in mice treated with anti-TIGIT+anti-PD-1 compared toanti-PD-1 monotherapy (p<0.0001). The combination ofanti-TIGIT+anti-PD-1 achieved synergistic anti-tumor efficacy that wasmore than the additive effect of both monotherapy treatments (p=0.02).The combination of anti-TIGIT (at 200 ug) and anti-PD1 antibodiesresulted in 7/8 mice showing a complete response. The anti-tumorefficacy was maintained with combination of anti-PD1 and lower doses ofanti-TIGIT that achieve complete response for 8/8 mice when anti-TIGITantibody was decreased to 60 μg and 5/8 mice when anti-TIGIT antibodywas decreased further to 20 μg (FIG. 16C). These data demonstrate thesignificant anti-tumor efficacy of anti-TIGIT therapy in monotherapy(p<0.0001) or in combination with an anti-PD1 antibody (p<0.0001) fortreatment of pre-established tumours.

Example 13: Isotype-Dependent Anti-Tumour Activity of Anti-TIGITAntagonistic Antibody in Monotherapy and Combination with Anti-PD1Antibodies in Mouse Model

For this experiment, anti-TIGIT clone 26493 was produced in mammaliancells on a mouse IgG2a and mouse IgG1 isotype. Female Balb/c mice of 8weeks were inoculated with 500.000 CT26 colon cancer cells (ATCC®CRL-2638™) subcutaneously. On day 10 after inoculation, when tumorvolumes were on average around 100 mm3, mice were randomized intreatment groups with equal tumor volume (n=10 per group). Forevaluation of monotherapy, mice were treated with 200 μg of anti-TIGITor isotype control (mIgG2a, BioXcell) by intraperitoneal injections onday 10, day 13 and day 16. For evaluation of combination with anti-PD-1,mice were treated with 200 μg of anti-PD-1 (RMP1-14, BioXcell) and 200μg of isotype control (mIgG2a, BioXcell) or by combination of 200 μg ofanti-PD-1 (RMP1-14, BioXcell) and 200 μg of anti-TIGIT byintraperitoneal injections on day 10, day 13 and day 16. Tumor growthwas monitored and tumor volumes were measured with electronic calipersthree times a week from day 10 until day 33. Mice were sacrificed whentumor volume exceeded 2000 mm3.

FIG. 17A shows median tumor growth curves per group as well asindividual growth curves for monotherapy with anti-TIGIT antibody andFIG. 17B for combination therapy with anti-TIGIT and anti-PD1antibodies. Both in monotherapy and in combination with anti-PD-1,treatment with anti-TIGIT antibody resulted in significant anti-tumorefficacy when administered as a mouse IgG2a isotype (p=0.0001 andp=0.009 respectively). However, no anti-tumor efficacy could be observedwith anti-TIGIT as a mouse IgG1 isotype, suggesting that interaction ofFc receptor with mIgG2a is important for the anti-tumor activity ofanti-TIGIT antagonistic antibodies in the murine CT26 model. These datademonstrate the isotype-dependent anti-tumor efficacy of anti-TIGITtherapy in monotherapy or combination for treatment of pre-establishedtumours.

Example 14: Characterization of the Mechanism of Action of In VivoAnti-Tumour Activity of Anti-TIGIT Antagonistic Antibody

A. Flow Cytometry Analysis of Spleen and Tumor

To investigate the in vivo mode of action of antagonistic anti-TIGITantibody, tumours were analysed by flow cytometry for the immune cellinfiltrate following treatment with anti-TIGIT antibody 26493 (IgG2a),in monotherapy and in combination with anti-PD-1. Mice were inoculatedand treated as described in example 12. Two days after the secondtreatment, mice (8 mice per group) were sacrificed and tumoursharvested. Tumours were dissociated with a tumour dissociation kit(Miltenyi Biotec). For direct ex-vivo staining, cells were stained withanti-CD45, anti-CD4, anti-CD8 and anti-FoxP3 (all from eBioscience)after staining with a viability dye (Molecular Probes, L34955) andFc-block. For ex vivo stimulation, cells were incubated with cellstimulation cocktail (eBioscience) and protein transport inhibitor(eBioscience) for 3 hours. This was followed by staining with anti-CD4and anti-CD8 antibodies and Fc-block. After fixation andpermeabilization with commercial buffers (IC fixation buffer andpermeabilization buffer), cells were stained with anti-IL-10 andanti-IFNg (all from eBioscience). In all the figures, the percentagechange compared to the relevant control group (isotype control formonotherapy, anti-PD-1 for combination) is shown, with a negative valuerepresenting a decrease and a positive value an increase compared to thecontrol group.

FIG. 18A shows that in vivo treatment of tumour with anti-TIGIT mIgG2aantibody results in a decrease in proportion of regulatory T cellswithin CD4+ TILs population of 28% compared to the control group, whichis not observed after treatment with anti-TIGIT mIgG1. This shows thatthere is a depletion of TIGIT+ Treg cells, possibly explaining thedifferential efficacy of the two isotypes as discussed in example 14.FIG. 18B shows that there is no depletion of CD8+ TILs, but instead asmall increase is observed for the two isotypes (a 17% increase comparedto control for mIgG1 and 16% for mIgG2a). These findings together resultin an increase of more than 50% of the CD8/Treg ratio in tumor treatedwith anti-TIGIT mIgG2a (FIG. 18C). Functionality of intratumoral T cellsis also improved for the group treated with anti-TIGIT mIgG2a antibody,with a strong increase in IFNg production of both CD4+(FIG. 18D) andCD8+ TILs (FIG. 18E). This resulted in a strong increase of the ratioIFN-g producing cells/IL-10 producing cells after ex vivo stimulation inthe CD4+ TILs/CD8+ population (FIG. 18F).

FIG. 18G shows that combining anti-TIGIT mIgG2a with anti-PD-1 resultsin regulatory T cells being decreased by 33% compared to anti-PD-1monotherapy. Again, for CD8+ T cells the opposite is true, with 22% and28% increase in CD8+ T cell infiltration, respectively for mIgG1 andmIgG2a isotypes, compared to anti-PD-1 monotherapy (FIG. 18H). Together,this results in more than two-fold increase in the CD8+ TILs to Tregratio in the tumor for the combination with anti-TIGIT mIgG2a (FIG.18I). Additionally, treatment with anti-TIGIT antibody mIgG2a combinedwith anti-PD-1 demonstrates a shift in Th1 versus Th2 phenotype forintratumor CD4+ T cells, with a marked increase in IFNg producing CD4cells (FIG. 18J) and a decrease in IL-10 producing CD4 cells (FIG. 18K).This resulted in a strong increase in IFNg/IL-10 producing cells afterex vivo stimulation in the CD4+ TILs population compared to mice treatedwith anti-PD-1 in monotherapy (FIG. 18L).

TABLE 11 Differentially expressed genes between anti- TIGIT mIgG2a andvehicle treated mice Gene symbol Log2 fold change Corrected p-value Ccr2−1.29 0.0000668 Prf1 1.79 0.0000668 Ctsg 2.13 0.0000668 Ctla4 1.720.00309 Gzmb 1.51 0.00309 Ccl2 0.56 0.0174 Il2ra 1.61 0.0174 Cd55 1.640.0213 Il2rb 0.872 0.0379 Cd274 0.982 0.0385 Klrg1 1.3 0.0402 Icos 1.260.0402 Il1rn 0.87 0.0402 Cx3cr1 −0.82 0.0428 C1ra 0.896 0.0428 Cd33−0.906 0.0479 Ccl4 0.886 0.0518

TABLE 12 Differentially expressed genes between anti-TIGIT mIgG2a +anti-PD-1 and anti-PD1 treated mice Gene symbol Log2 fold changeCorrected p-value Ctsg 2.34 0.0000375 Prf1 1.69 0.000255 Gzmb 1.710.000766 Cd55 2.08 0.00131 Entpd1 0.839 0.00131 Klrg1 1.76 0.00132 Itga10.874 0.0017 Ctla4 1.72 0.00173 Il2ra 1.82 0.00237 Itgb3 0.863 0.00237Slc11a1 0.849 0.00329 Cd36 1.44 0.0049 Cd180 0.899 0.00602 Icam1 0.8930.00802 Cd274 1.06 0.00993 Cd40 0.926 0.0113 Eomes 1.28 0.0113 Abcg10.869 0.0113 Ccr2 −0.781 0.0122 Thy1 0.868 0.0165 Ccl2 0.501 0.0203 Gbp51.12 0.0216 Icos 1.24 0.0263 Tgfbr2 0.458 0.0278 H2 K1 0.292 0.0307Sh2d1a 0.999 0.0307 Il2rb 0.808 0.0307 Selplg 0.64 0.031 Bst1 0.7020.0317 Cd247 1 0.032 Irf8 0.699 0.0365 Il21r 0.899 0.0392 Gbp2b 1.110.0392 Stat1 0.865 0.0427 C4b 0.922 0.0428 Abca1 0.537 0.044 Trem2 0.4820.0454

B. Transcriptomics Analysis of Tumor by NanoString

To investigate the in vivo mode of action of anti-TIGIT antibody, theimmune cell infiltrate of tumours treated with anti-TIGIT, inmonotherapy and in combination with anti-PD-1, was analysed bytranscriptomic analysis (Nanostring). Mice were inoculated and treatedas described in Example 12. Two days after the third treatment withanti-TIGIT and/or anti-PD1 antibodies, mice were sacrificed and tumorsharvested. RNA was extracted and the expression of a selection of 770genes involved in cancer immunology was directly quantified with thenCounter technology (PanCancer Immune Profiling panel, Nanostring;performed by VIB Nucleomics Core). Data were analyzed with nSolversoftware (Nanostring).

FIG. 19A shows a volcano plot of the genes that are differentiallyregulated between vehicle treated mice and anti-TIGIT mIgG2a treatedmice. Highly statistically significant genes fall at the top of theplot, and highly differentially expressed genes fall to either side(left: downregulated in anti-TIGIT treated mice, right: upregulated inanti-TIGIT treated mice). Examples of highly upregulated genes includeperforin, granzyme B and CTLA-4. The solid line represents anon-corrected p-value of 0.01, the dotted line a corrected p-value of0.05 (Benjamini-Hochberg correction). Table 11 and Table 12 show thegenes that were significantly differentially expressed for anti-TIGITmIgG2a compared with vehicle and aPD-1+anti-TIGIT mIgG2a versus anti-PD1respectively. When multiple genes were summarized in scores forfunctional subsets of immune cells, the most striking finding was anincrease in cytotoxic cell and CD8+ T cell score (FIG. 19B). The samechanges were present in mice treated with anti-PD-1+anti-TIGIT mIgG2acompared to anti-PD-1 alone. No changes were observed in mice treatedwith anti-TIGIT mIgG1, in monotherapy or in combination with anti-PD-1.

Altogether, these results demonstrate that the anti-tumour efficacyobserved after in vivo treatment with anti-TIGIT antibody is mediated bya decreased Treg infiltrate in the tumour while CD8+ effector T cellpopulation is increased. In addition, effector function of CD4+ and CD8+TILs are increased as shown by the higher proportion of IFNg producingcells, the shift towards Th1 response and the increased expression ofgenes important for T cell cytotoxic functions.

Example 15: Antibody Dependent Cellular Toxicity (ADCC) Activity Inducedby Anti-TIGIT Antagonistic Antibodies

A. In Vitro ADCC on Human PBMC from Healthy Donors

Isolated PBMCs from healthy human donors were resuspended in completeRPMI medium (supplemented with 10% FBS heat inactivated+50 UPenicilin+50 U Streptomycin, and supplemented with 200 IU IL-2/ml).2.5×105 human PBMCs were distributed per well in 96 U well plate.Anti-human TIGIT antibody clone 26452 produced in mammalian cells orIgG1 isotype control (Biolegend, 403102) were added at a finalconcentration of 66.6; 0.66 and 0,006 nM to each corresponding well.Cells were incubated for 20 h at 37° C. with 5% CO2. Then cells werecollected and stained with the following antibody panel: LVD efluor 520(eBioscience 65-0867-14), ant-TCRab-PercP-Cy5.5 (Clone IP26, Biolegend306723), anti-CD4-BV510 (Clone SK3, BD Horizon 562970), anti-CD8-APC-Cy7(Clone SK1, Biolegend 344714), anti-CD25-BV605 (Clone 2A3, Biolegend562660), anti-CD127-APC (A019D5, Biolegend 351316), anti-CCR7-BV421(Clone G043H7, Biolegend 353207) and anti-CD45RO-PE-Cy7 (Clone UCHL1,Biolegend 304229). Results are presented on gated live cells. CD45+CD4+or CD45+CD8+ represent the total CD4+ or CD8+ T cells. CD45+RO+CD4+ orCD45+RO+CD8+ cells represent the memory CD4+ or CD8+ T cells whileCD25hiCD127lowCD4+ represent Treg cells. The proportion of TIGIT+ cellson gated Tregs is higher than on gated memory CD8+ T cells and CD4+ Tcells, as shown in FIG. 20A.

Absolute quantification is done using AccuCheck Counting beads (Lifetechnologies) following manufacturer's specifications. After calculationof absolute cell numbers per μl, % of specific lysis is calculated usingthe following formula=(1−(absolute number of cells per μl on 26452 TIGITantibody treated sample/average of triplicate of no antibodytreatment))×100. As shown in FIG. 20B anti-TIGIT 26452 hIgG1 antibodytriggers higher specific lysis on Tregs (62.22%) than on total CD8+ Tcells (12.2%) or total CD4+ T cells (16.36%).

B. Ex-Vivo ADCC on Mouse Tumor

To confirm that anti-TIGIT mouse IgG2a antibody can deplete TIGIT+regulatory T cells, an ex-vivo ADCC assay was set-up. Female Balb/c miceof 8 weeks were inoculated with 500.000 CT26 colon cancer cells (ATCC®CRL-2638™) subcutaneously. Three weeks after inoculation, tumors wereharvested and dissociated with a tumor dissociation kit (MiltenyiBiotec). The single cell suspension was incubated with 133 nM anti-TIGITantibody 26493 (mIgG1 or mIgG2a isotype) for 20 h (1 million cells/200Win RPMI+10% FBS). After 20 h, cells were stained with anti-CD4,anti-TIGIT, anti-CD8 and anti-FoxP3 antibodies (all from eBioscience)after staining with a viability dye (Molecular Probes, L34955) andFc-block.

FIG. 21 shows the % decrease in absolute TIGIT+ cell counts compared totreatment with isotype control for the different TIGIT+ immune subsets.The strongest decrease after anti-TIGIT mIgG2a antibody treatment isevident in regulatory T cells (around 40% decrease), suggesting thatthese cells are more susceptible to ADCC than conventional CD4+ or CD8+T cells.

Overall, these results demonstrate the efficacy of anti-TIGIT hIgG1 ormIgG2a to deplete TIGIT+ immune cells with a stronger activitydemonstrated on Treg population.

Example 16: Immunogenicity Prediction Using in Silico Analysis

Immunogenic potential of clones 29494 and 29489 as well as its variant31282 was assessed by in silico prediction using EpiMatrix Protein Score(De Groot et al. (2009) Clinical Immunol. 131:189). To complete theanalysis, the input sequences were parsed into overlapping 9-mer framesand each frame was evaluated with respect to a panel of eight commonClass II HLA alleles. These alleles are “super-types”. Each one isfunctionally equivalent to, or nearly equivalent to, many additional“family member” alleles. Taken collectively, these eight super-typealleles, along with their respective family members, “cover” well over95% of the human population (Southwood et al. (1998) J. Immunol160:3363). Each frame-by-allele “assessment” is a statement aboutpredicted HLA binding affinity. EpiMatrix assessment scores range fromapproximately −3 to +3 and are normally distributed. EpiMatrixassessment scores above 1.64 are defined as “hits”; that is to saypotentially immunogenic and worthy of further consideration.

All other factors being equal, the more HLA ligands (i.e. EpiMatrixhits) contained in a given protein, the more likely that protein is toinduce an immune response. The EpiMatrix Protein Score is the differencebetween the number of predicted T cell epitopes expected to be found ina protein of a given size and the number of putative epitopes predictedby the EpiMatrix System. The EpiMatrix Protein Score is correlated withobserved immunogenicity. EpiMatrix Protein Scores are “normalized” andcan be plotted on a standardized scale. The EpiMatrix Protein Score ofan “average” protein is zero. EpiMatrix Protein Scores above zeroindicate the presence of excess MHC ligands and denote a higherpotential for immunogenicity while scores below zero indicate thepresence of fewer potential MHC ligands than expected and a lowerpotential for immunogenicity. Proteins scoring above +20 are consideredto have a significant immunogenic potential.

Adjusting for the Presence of Regulatory T Cell Epitopes.

Antibodies are unique proteins in that the amino acid sequences of theirvariable domains, especially their Complementarity Determining Regions(CDRs), can vary to an extraordinary extent. It is this variability thatallows antibodies to recognize a wide variety of antigens. However, therecombination and mutation events that control antibody maturation canalso produce new or neo-T cell epitopes. These neo-epitopes can appearto be “foreign” to circulating T cells. The presence of neo-epitopes inantibody sequences can lead to the formation of a human-anti-humanantibody response; also known as the HAHA response or ADA(Anti-Drug-Antibodies).

Regulatory T cells play an important role in suppressing immuneresponses to fully human proteins in the periphery, including thosecontaining mutated and/or highly variable sequences such as antibodyCDRs. Regulatory T cells are engaged and activated by regulatory T cellepitopes. The inherent risk associated with the presence of neo-epitopesin antibody sequences appears to be balanced by the presence ofnaturally occurring regulatory T cell epitopes.

By screening the sequences of many human antibody isolates, EpiVax hasidentified several highly conserved HLA ligands which are believed tohave a regulatory potential. Experimental evidence suggests many ofthese peptides are, in fact, actively tolerogenic in most subjects.These highly conserved, regulatory, and promiscuous T cell epitopes arenow known as Tregitopes (De Groot et al. (2008) Blood 112:3303)

In many cases, the immunogenic potential of neo-epitopes contained inhumanized antibodies can be effectively controlled in the presence ofsignificant numbers of Tregitopes. For the purposes of antibodyimmunogenicity analysis, EpiVax has developed a Tregitope-adjustedEpiMatrix Score and corresponding prediction of anti-therapeuticantibody response. To calculate the Tregitope-adjusted EpiMatrix Score,the scores of the Tregitopes are deducted from the EpiMatrix ProteinScore. The Tregitope-adjusted scores have been shown to be wellcorrelated with observed clinical immune response for a set of 23commercial antibodies (De Groot et al. (2009) Clinical Immunol.131:189).

Clones 29489, 29494 and 31282 antibody sequences score on the low end ofEpiMatrix scale, indicating limited potential for immunogenicity.Regression analysis of licensed monoclonal antibodies predicts ADAresponse in ˜0% of exposed patients for antibody clone 29489 and 31282.For clone 29494, analysis predicts ADA response in 2.78% of exposedpatients for the baseline VH sequence, and 2.88% for the variant VHsequence. Data are summarized in Table 13, below.

TABLE 13 EpiMatrix and Tregitope adjusted EpiMatrix Scores Tregitopeadjusted Input EpiMatrix EpiMatrix EpiMatrix Sequence Length AssessmentsHits Score Score 29489_VH 121 904 40 −19.41 −47.26 29489_VL 108 800 39−17.58 −51.75 31282_VH 121 904 40 −19.41 −47.26 31282_VL 108 800 39−17.58 −51.75 29494_VH 125 936 54 2.68 −7.18 29494_VL 107 792 40 −12.2−38.83

Example 17: Affinity Determination for Binding of Anti-TIGIT Clones toRecombinant Human TIGIT Protein

Antibody 31282 was compared against anti-TIGIT antibody clones describedin other patent applications. Specifically, 31282 was compared with:4.1D3.Q1E (also referred to as 4.1D3, from WO2017/053748); 22G2 (fromWO2016106302); 3106 (from WO2016/028656); 313M2 (from WO2016/191643);and TIG1 (from WO2017/152088). The references and sequences of thecompared antibody clones are shown in Table 14 below:

TABLE 14Sequences of VH and VL domains of comparative anti-TIGIT antibodiesa-TIGIT clone Reference Sequence 4.1D3.Q1E VH: SEQ ID NO: 34 ofVH sequence: WO2017/053748 EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRVL: SEQ ID NO: 36 of QSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNWO2017/053748 QFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO: 343 herein) VL sequence:DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK (SEQ ID NO: 344 herein) 22G2VH: SEQ ID NO: 7 of VH sequence: WO2016/106302QVHLQESGPGLVKPSETLSLTCTVSGGSVSSGIYYWSWIR VL: SEQIDNO:9ofQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQF WO2016/106302SLKLSSVTAADTAVYYCARDYYVSGNYYNVDYYFFGVDVWGQGTTVTVSS (SEQ ID NO: 345 herein) VL sequence:EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRSNWPPLFTFGPGTKVDIK(SEQ ID NO: 346 herein) 31C6 VH: SEQ ID NO: 127 of VH sequence:(MEB125.31C WO2016/028656 EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVR6.A1.205 VL: SEQ ID NO: 130 of QAPGQGLEWIGYIDPYNDGAKYAQKFQGRVTLTSDKSTSVH4/VL1) WO2016/028656 TVYMELSSLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSS (SEQ ID NO: 347 herein) VL sequence:DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQHHFGSPLTFGQGTRLEIK(SEQ ID NO: 348 herein) 313M32 VH: SEQ ID NO: 67 of VH sequence:WO2016/191643 QVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWNWIRQPVL: SEQ ID NO: 68 of PGKGLEWIGYISYSGSTSYNPSLRSRVTISRDTSKNQFFLKWO2016/191643 LSSVTAADTAVYYCARRQVGLGFAYWGQGTLVTVSS(SEQ ID NO: 349 herein) VL sequence:DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPWTFG (SEQ ID NO: 350 herein) TIG1 VH: SEQ ID NO: 10 ofVH sequence: WO2017/152088 DVQLVESGGGLVQPGGSRKLSCAASGFTFSNFGMHWVRQVL: SEQ ID NO: 14 of APEKGLEWVAFISSGSSSIYYADTVKGRFTISRDNPKNTLFWO2017/152088 LQMTSLRSEDTAMYYCARMRLDYYAMDYWGQGTSVTVSS (SEQ ID NO: 351 herein) VL sequence:DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPED FAMYYCQQHNEYPWTFGGGTKLEIK(SEQ ID NO: 352 herein)

A. Production in Mammalian Cells

In order to produce sufficient amounts of selected a-TIGIT clones forfurther characterization, DNA vectors coding for specific antibodyclones (clones 31282_up, 4.1D3, 22G2, 3106, 313M32 and TIG1) weregenerated and transduced into HEK cells for production of human IgG1isotype. Human codon optimized synthetic DNA fragments for antibodyvariable domains were ordered at Geneart. Variable domain sequences wereseamlessly ligated into pUPE expression vectors containing the mouseIgKappa signal sequence and constant regions of the respective antibodyclass. Expression vectors were verified by restriction analysis and DNAsequencing. For transient transfection Endotoxin free DNA maxipreps(Sigma) were produced and heavy and light chain vectors wereco-transfected to HEK293EBNA1 cells, in Freestyle medium(ThermoFisherScientific), according to established protocols. Primatone(0.55% final volume) was added 24 hours post-transfection. Conditionedmedium was harvested 6 days post transfection. Antibodies were purifiedbatch wise by Mabselect sureLX (GE Healthcare) affinity chromatography.Bound antibodies were washed in 2 steps with PBS containing 1M NaCl andPBS. Antibodies were eluted with 20 mM Citrate 150 mM NaCl pH3 andneutralized to approximately pH7 with ⅙ volume of 1M K2HPO4/KH2PO4 pH8.

Next the antibodies were further purified by gel-filtration using aSuperdex200 column, equilibrated in PBS. Fractions were analysed byNuPAGE and antibody containing fractions were pooled. The final productswere sterilized over a 0.22 μM syringe filter. The product was analysedby NuPAGE and endotoxin levels were measured by LAL-assay.

Additionally, clone 31282 was also produced in CHO-K1 cell as follow(clone 31282_wu) on IgG1 or IgG4 isotype. DNA vectors coding for theantibodies were constructed and transfected into CHO-K1 cells. CHO codonoptimized DNA fragments for antibody variable domains were synthesized,and ligated into expression vectors containing the signal sequence andconstant regions of the respective antibody class. Expression vectorswere verified by restriction analysis and DNA sequencing. Heavy andlight chain vectors were co-transfected to CHO-K1 cells byelectroporation (Bio-rad) according to established protocols. Thetransfected cultures were scaled up and inoculated into fed-batchcultures. Conditioned medium was harvested after 14 days of fed-batchcultures.

Harvested cell culture was firstly clarified by two stages of depthfiltration with DOHC and A1HC (Millipore) connected in series. Then, theclarified harvest was firstly purified by affinity chromatography withMabSelect SuRe (GE Healthcare). Bound antibodies were washed in 2 stepswith 50 mM NaAc-HAc (pH 5.5) containing 1 M NaCl and 50 mM NaAc-HAc (pH5.5). Antibodies were then eluted with 50 mM NaAc-HAc (pH 3.5) andneutralized to approximately pH 5.5 with 1 M Tris-HCl (pH 9.0).

Next the neutralized intermediate was further polished by anion exchangechromatography (AEX) using POROS HQ50 (Life Tech) in flow-through mode.The column was equilibrated by 50 mM NaAc-HAc (pH 5.5) before loading.AEX flowthrough collected during loading and recovering step was furtherpolished by cation exchange chromatography (CEX) in bind-elute modeusing POROS XS (Life Tech.). The CEX column was equilibrated in 50 mMNaAc-HAc (pH 5.5), and the antibodies were eluted out by linear gradientelution (LGE) to reach 50 mM NaAc-HAc (pH 5.5) containing 0.5 M NaCl in10 CV. The final ultrafiltration and dia-filtration (UF/DF) usingPellicon 3, ultracel 30 kD, type A (Millipore) was performed toconcentrate the CEX eluate and exchange buffer into 20 mM His-HCl (pH5.5). Afterwards, Polysorbate 80 (PS80) and sucrose was added into thedia-filtrated sample to obtain the final product of which theconcentration was proximately 20 g/L, in the buffer of 20 mM His-HCl,0.01% (w/w) PS 80, and 9% (w/v) sucrose (pH 5.5). The product had gonethrough all PQA tests. The SEC purity, Endotoxin level and othercriteria had all met the requirement.

B. Biacore Measurement

Biosensor analysis was conducted at 25° C. in a HBS-EP buffer system (10mM HEPES pH 7.3, 150 mM NaCl, 3 mM EDTA, 0.05% Tween20) using BiacoreT200 technology, CM5 sensor chip (run at Novalix, France). The samplehotel was maintained at 8° C. Goat anti-human IgG capture antibody (Fcγfragment specific, Jackson ImmunoResearch Laboratories) was immobilized(10000 RU) to both flow cells of the sensor chip using standard aminecoupling chemistry. This surface type provided a format for reproduciblycapturing fresh analysis antibody after each regeneration step. Flowcell 2 was used to analyse captured antibody while flow cell 1 was usedas a reference flow cell. 6 different antigen concentrations rangingfrom 30 to 0.123 nM were prepared in running buffer. Each of the antigensample concentrations were run as a single replicate, except 3.33 nM runin duplicate. Two blank (buffer) injections also were run and used toassess and subtract system artefacts. The association (300 s) anddissociation (600 s) phases for all antigen concentrations wereperformed at a flow rate of 30 μl/min. The surface was regenerated withthree sequential injections (15 s, 15 s and 60 s) of 10 mM glycine-HCl,pH 1.5. The obtained sensorgrams were fitted globally to a 1:1 model(assuming the same kinetic values for all applied concentrations).Affinity was also determined from steady state for clone 313M32 as 1:1kinetic model fitting was not reliable, showing equilibrium with humanTIGIT at the end of the association time. Results obtained for thedifferent a-TIGIT clones are reported in Table 15.

TABLE 15 Kinetic and affinity evaluation Kinetic model 1:1 BindingSteady State Model Ka Kd K_(d) Rmax K_(d) Rmax Clone (1/s) (1/s) (nM)(RU) (nM) (RU) 31282_wu 3.86⁺⁰⁶ 4.62⁻⁰⁴ 0.120 16.7 31282_up 3.70⁺⁰⁶4.75⁻⁰⁴ 0.128 15.3 4.1D3 1.07⁺⁰⁶ 4.72⁻⁰⁵ 0.044 14.4 22G2 2.51⁺⁰⁶ 1.78⁻⁰⁴0.071 11.1 31C6 3.10⁺⁰⁶ 2.09⁻⁰⁴ 0.067 16.7 313M32 na na na na 10.1 17.2TIG1 5.24⁺⁰⁶ 1.31⁻⁰² 2.49  11.1

Example 18: Cellular Binding of Anti-TIGIT Antagonistic Antibodies

A. Binding of Anti-TIGIT Clones to Jurkat-hTIGIT

The affinity of human anti-TIGIT antibodies has been measured usingJurkat E6.1 cells transduced with human-TIGIT (Jurkat hTIGIT). Toanalyse the affinity of the selected antibodies for hTIGIT, 105 cellswere distributed per well and incubated with decreasing concentration(8; 4; 2; 1; 0.5; 0.25; 0.125; 0.062; 0.031; 0.016; 8×10⁻³ and 4×10⁻³nM) of various anti-TIGIT antagonist antibody clones (FIG. 2 ).Antibodies were incubated with the cells for 20 min at 4° C. in FACSbuffer. After washing, cells were incubated with anti-human Ig (Fc gammaspecific)-PE (eBioscience, 12-4998-82, at 2.5 μg/ml) for 20 min on iceand washed twice. Fluorescence intensity was analysed using LSR BDFortessa and cell binding was recorded as the median fluorescenceintensity of PE in cells expressing TIGIT at their surface.

The half-maximal concentration of binding (EC50) to Jurkat-hTIGIT wascalculated using a four-variable curve-fit equation in Prism. Theresults are illustrated in FIG. 22A and the values summarized in theTable 16 below. EC50 values for binding Jurkat-hTIGIT are very close forclone 31282 with no marked difference between antibody produced in HEKcells (31282 up, 0.13 nM) or in CHO-K1 cells (31282-wu, 0.10 nM). Clone3106 and TIG1 also show EC50 values below 0.2 nM while affinity forother clones (4.1D3, 22G2 and 313M32) is lower and results show EC50values ranging from 0.267 to 0.445 nM. Results demonstrate a strongbinding to membrane expressed human TIGIT in an engineered system foranti-TIGIT clones 31282, 3106 and TIG1 while other clones have a loweraffinity.

TABLE 16 EC₅₀ data and comparison of different a- TIGIT clones forbinding to Jurkat-hTIGIT Fold change of EC₅₀ EC₅₀ binding to over EC₅₀of best Clone Jurkat-hTIGIT (in nM) clone (31282_wu) 31282_wu 0.10 131282_up 0.13 1.3 313M32 0.44 4.2 4.1D3 0.27 2.5 22G2 0.32 3.0 31C6 0.131.2 TIG1 0.17 1.6

B. Binding of Anti-TIGIT Clones to Primary CD8⁺ T Cells from HealthyHuman PBMCs

Isolated human PBMCs from healthy volunteers were analysed for bindingby antagonist anti-TIGIT antibodies. Cells were distributed at 1×105cells per well. Cells were incubated with anti-CD16 (Clone 3G8,BioLegend 302002), CD32 (Clone FLI8.26, BD Bioscience 557333) and CD64(BD Bioscience 555525) at room temperature for 10 min, and the indicatedanti-human TIGIT antibody clones were directly added at a finalconcentration of 8; 4; 2; 1; 0.5; 0.25; 0.125; 0.062; 0.031; 0.016;8×10-3 and 4×10−3 nM in FACS buffer and incubated for 20 min at 4° C.After washing, cells were incubated with anti-human Ig (Fc gammaspecific)-PE (eBioscience, 12-4998-82, at 2.5 μg/ml) for 20 min at 4° C.Then, cells were washed and incubated with the following antibodies andLVD mix: anti-CD4-PercP-Cy5.5 (clone A161A1, BioLegend 357414);anti-CD8-BV510 (clone SK1, BD Bioscience 563919) and LVD efluor 660(eBioscience 65-0864-18).

The EC50 values for binding to CD8+ human primary T cells werecalculated using the MFI signal on living TIGIT+CD8+ T cells. Theresults are illustrated in FIG. 22B and the EC50 concentrationssummarized in the Table 17 below. EC50 values for binding human primaryCD8+ T cells are very close for clone 31282 with no marked differencebetween antibody produced in HEK cells (31282_up, 0.21 nM) or in CHO-K1cells (31282-wu, 0.19 nM). Comparison between the different clones ofantagonist a-TIGIT antibodies show the best EC50 value for binding onhuman primary CD8+ T cells for clone 31282 wu (0.19 nM) and clone31282_up (0.21 nM). Clones 3106 and TIG1 show a difference in EC50 of 2fold while clone 22G2, 313M32 and 4.1D3 differs by a factor of 6.1 to9.7 fold. Overall, 31282 wu and 31282_up show the best binding tomembrane expressed TIGIT on human primary CD8+ T cells.

TABLE 17 EC₅₀ data and comparison of different a-TIGIT clones forbinding to Human primary CD8⁺ T cells Fold change of EC₅₀ EC₅₀concentration for binding over EC₅₀ of best Clone to CD8⁺ T cells (innM) clone (31282_wu) 31282_wu 0.19 1 31282_up 0.21 1.1 313M32 1.45 7.54.1D3 1.88 9.7 22G2 1.17 6.1 31C6 0.39 2.0 TIG1 0.38 2.0

C. Binding of Anti-TIGIT Clones to Primary CD8⁺ T Cells from CancerPatients PBMCs

Isolated human PBMCs from cancer patients were analysed for binding bydifferent antagonist anti-TIGIT antibody clones. Cells were distributedat 1×105 cells per well. Cells were incubated with anti-CD16 (Clone 3G8,BioLegend 302002), CD32 (Clone FLI8.26, BD Bioscience 557333) and CD64(BD Bioscience 555525) at room temperature for 10 min, and the indicatedanti-human TIGIT antibodies were directly added at a final concentrationof: 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.062 and 0.031 nM in FACS buffer andincubated for 20 min at 4° C. After washing, cells were incubated withanti-human Ig (Fc gamma specific)-PE (eBioscience, 12-4998-82, at 2.5μg/ml) for 20 min at 4° C. Then, cells were washed and incubated withthe following antibodies and life viability dye (LVD) mix:anti-CD4-PercP-Cy5.5 (clone A161A1, BioLegend 357414); anti-CD8-BV510(clone SK1, BD Bioscience 563919) and LVD efluor 520 (eBioscience65-0867-14). Cells were washed and fixed and surface staining wasquantified using BD LSR Fortessa. Flow cytometry data was analysed usingFlowJo V10.1. TIGIT MFI on gated LVD-TIGIT+CD8+ cells was used tocalculate EC50 values. Nonlinear regression curves are shown on FIG. 22Cand the values summarized in Table 18 below.

Clones 31282_wu and 31282_up show very close EC50 value for binding onCD8+ T cells from cancer patients with concentration of 0.14 and 0.12nM, respectively. The rest of the clones show lower affinity with clone3106, TIG1 and 22G2 showing a 1.5, 2.7 and 3.1 fold lower affinity,respectively. Measured EC50 value for clone 313M32 is 8.3 fold lowercompared to clone 31282_up. Clone 4.1D3 shows the lowest affinity,binding with a difference of 9.5 fold to the best clone tested.

TABLE 18 EC₅₀ data and comparison of different a-TIGIT clones forbinding to Human primary CD8⁺ T cells from cancer patients Fold changeof EC₅₀ EC₅₀ value for CD8⁺ over EC₅₀ of best Clone T cells binding (innM) clone (31282) 31282_wu 0.14 1.2 31282_up 0.12 1.0 313M32 1.0 8.34.1D3 1.15 9.5 22G2 0.37 3.1 31C6 0.18 1.5 TIG1 0.33 2.7

Example 19: Competition Assay Between Anti-TIGIT Antagonist AntibodyClones and TIGIT Natural Ligand (CD155)

Jurkat cells overexpressing human TIGIT (Jurkat-hTIGIT) were collectedand distributed at 5·10⁴ cells/well and incubated with anti-human TIGITantibodies at the following concentrations: 133.33; 42.20; 13.33; 4.22;1.33; 0.422; 0.133; 0.042; 0.0133; 4.2×10⁻³; 1.3×10⁻³; 4.2×10⁻⁴;1.3×10⁻⁴; 4.2×10⁻⁵ nM in complete medium during 45 min at 37° C. Excessof antibody was washed, and then the cells were incubated with CD155-Hisat 15 μg/ml (Creative Biomart, PVR-3141H) for 45 min at 37° C. Then,bound CD155-His was detected using anti-His tag-PE (Biolegend, 362603,at 2 μl per test), incubated for 30 min at 4° C. Cells were analysed byFACS using BD LSRFortessa and the half concentration (IC50) thatprevents CD155 binding was calculated based on the median fluorescenceintensity of PE in total cells.

The results are illustrated in FIG. 23 and the values summarized in theTable 19 below. Anti-TIGIT clones 31282_wu and 31282 up show the bestIC₅₀ values for CD155 competition on Jurkat cells engineered to expresshTIGIT with concentration of 0.05 and 0.04 nM respectively. Other clones(4.1D3, 22G2, 3106, TIG1) have IC50 values between 0.07 and 0.09 nMwhile clone 313M32 clone competes with CD155 for binding to TIGIT with amuch lower efficiency (0.65 nM).

TABLE 19 IC₅₀ data and comparison of different a-TIGIT clones for CD155competition on human TIGIT Fold change of IC₅₀ IC₅₀ of CD155 competitionover IC₅₀ of best Clone for TIGIT binding (in nM) clone (31282_up)31282_wu 0.05 1.3 31282_up 0.04 1 313M32 0.65 16.7 4.1D3 0.07 1.9 22G20.09 2.2 31C6 0.07 1.7 TIG1 0.06 1.6

Example 20: Functional Characterization of Antagonistic Anti-TIGITClones

A. TIGIT Functional Assay with Jurkat-hTIGIT Cells

To characterize the functional consequence of blocking human TIGITreceptor, we co-cultured Jurkat cells, that express hTIGIT and aluciferase reporter activated upon TCR engagement (Thaw-and-Use TIGITEffector cells from Promega), with CHO-K1 cell line engineered toexpress human PVR/CD155 and TCR activator (Thaw-and-Use CD155aAPC/CHO-K1 from Promega). The activation of TIGIT-overexpressing Jurkatcells can be induced by contact with CD155-expressing CHO-K1 cells uponTCR engagement on Jurkat cells and can be increased in presence ofantagonist anti-TIGIT antibody. To compare the potency of the differenta-TIGIT clones to increase Jurkat cell activation, the experiment wasconducted in presence of increasing antibody concentrations and the EC50values were calculated.

CD155 aAPC/CHO-K1 (Promega, CS198811) cells were seeded according tomanufacturer's recommendations and incubated at 37° C., 5% CO2 incubator0/N. The next day, TIGIT Effector cells (Promega, CS198811) were addedaccording to manufacturer's recommendations to the CD155 aAPC/CHO-K1cell plates containing fresh full medium with anti-TIGIT antibody atincreasing concentrations (0.03; 0.11; 0.33; 1.06; 3.34; 10.56; 33.38;105.49; and 333 nM) and incubated at 37° C., 5% CO2 during 6 hours.After the 6 hours of incubation, activation of TIGIT Effector cell wasassessed by measuring the luciferase activity by using Bio-Glo™Luciferase Assay System (Promega, G7941).

As shown in FIG. 24A and summarized in Table 20, anti-TIGIT antibody31282 has the best efficacy in term of EC50 value and maximum inductionof luciferase signal in the assay. Activity observed for clone producedin HEK (31282 up) or CHO-K1 (31282_wu) cells is comparable with amaximum luciferase signal that is 8 folds higher than control isotype(Bioexcell, BE0297) and with an EC50 concentration measured at 3.3 nMand 3.5 nM respectively. By way of comparison, clones 4.1D3, 22G2 and3106 have a maximum activity between 5.3 and 6.7 fold over isotypecontrol, associated with an EC50 between 5 and 10 nM. EC50 values forclone 313M32 and TIG1 could not be determined due to a low activity andpoor fitting of the curves at the concentrations tested (FIG. 24A).

TABLE 20 EC₅₀ data and comparison of different a-TIGIT clones forfunctional activity on Jurkat-hTIGIT cells Induction over Fold change ofEC₅₀ Isotype control EC₅₀ over EC₅₀ of best Clone name (fold change)(nM) clone (31282_up) 31282_wu 8.4 3.5 1.1 31282_up 8.0 3.3 1   313M32 /P.F. / 4.1D3 5.8 10.3  3.1 22G2 5.3 5.2 1.6 31C6 6.7 5.3 1.6 TIG1 / P.F./ P.F.: poor fit

B. TIGIT Functional Assay on Human Primary CD8⁺ T Cells from HealthyVolunteers

To characterize the functional consequence of blocking human TIGITreceptor, we co-cultured human primary CD8+ T cells from PBMC of healthyhuman donors with CHO-K1 cell line engineered to express human PVR/CD155and to activate human T cells. We observed that the release of IFNg byCD8+ T cells in presence of engineered CD155-expressing CHO-K1 cellscould be increased by blocking hTIGIT with anti-TIGIT antagonisticantibodies. To compare the potency of these antibodies to increase IFNgrelease, the experiment was conducted in the presence of increasingantibody concentrations and the EC50 values were calculated.

CD155 aAPC/CHO-K1 (Promega, CS198811) cells were seeded in U-bottom96-well plates according to manufacturer's recommendations and incubatedat 37° C., 5% CO2 incubator 0/N. The next day, CD8+ T cells werepurified according to manufacturer's recommendations by using negativeselection kit (Stemcell Technologies, 17953) from frozen humanperipheral blood mononuclear cells isolated from total blood of healthydonors (Immunehealth). Purified CD8 T cells and increasingconcentrations (0.011 nM. 0.033 nM, 0.11 nM, 0.33 nM, 1.06 nM, 3.3 nM,10.6 nM, 33.3 nM and 105.5 nM) of antibodies were then added to CD155aAPC/CHO-K1 (100,000 CD8 T cells/100W of full medium containingantibody) and incubated at 37° C., 5% CO2 during 5 days. Finally, IFNgconcentrations were assessed in cell supernatant using an ELISA assay(Affymetrix eBioscience, 88-7316-86) that was run according tomanufacturer's recommendations.

As shown in FIG. 24B and summarized in Table 21, a-TIGIT clone 31282 and4.1D3 display the best induction of IFNg secretion with a respective 2.7and 2.9 fold increase over isotype control antibody. Clone 31282 has thebest efficacy for induction of IFNg production in terms of EC50concentration, which was measured at 0.13 nM. Clone 3106 shows an EC50value 2.3 fold different while clone 22G2 and 4.1D3 are 3.1 and 10.8fold less potent than clone 31282. No value could be determined forclone 313M32 due to a low activity and poor fitting of the curves at theconcentrations tested (FIG. 24B).

TABLE 21 EC₅₀ data and comparison of different a-TIGIT clones forfunctional activity on human primary CD8⁺ T cells Induction over Foldchange of EC₅₀ over isotype control EC₅₀ over EC₅₀ of best Clone (foldchange) (nM) clone (31282_wu) 31282_wu 2.7 0.13 1 313M32 / P.F. / 4.1D32.9 1.43 10.8 22G2 1.5 0.41 3.1 31C6 1.6 0.30 2.3

C. TIGIT Functional Assay on Human Primary CD3⁺ T Cells from CancerPatients

To characterize the functional consequence of blocking human TIGITreceptor on T cells from cancer patients, human primary CD3+ T cellsfrom PBMC of a cancerous patient were co-cultured with a CHO-K1 cellline engineered to express human PVR/CD155 and to activate human T cells(CHO-TCR-CD155). We observed that the release of IFNg by CD3+ T cells inthe presence of engineered CD155-expressing CHO-K1 cells could beincreased by blocking hTIGIT with anti-TIGIT antagonist antibody 31282.

CD155 aAPC/CHO-K1 (Promega, CS198811) cells were seeded in U-bottom96-well plates according to manufacturer's recommendations and incubatedat 37° C., 5% CO2 incubator 0/N. The day after, CD3+ T cells werepurified according to manufacturer's recommendations by using negativeselection kit (Stemcell Technologies, 17951) from fresh human peripheralblood mononuclear cells isolated from total blood from a cancerouspatient (HNSCC) collected 24 h earlier (Biopartners). Purified CD3+ Tcells and 66.7 nM of antibodies were then added to CD155 aAPC/CHO-K1(100,000 CD3 T cells/100W of full medium containing antibody) andincubated at 37° C., 5% CO2 for 5 days. Finally, IFNg concentrationswere assessed in cell supernatant using an ELISA assay (AffymetrixeBioscience, 88-7316-86) that was run according to manufacturer'srecommendations.

As shown in FIG. 24C, antibody 31282 induced a strong functionalactivity to increase IFNg secretion, demonstrating the potential of thisa-TIGIT antibody to reactivate PBMC T cells from cancer patients.

D. a-TIGIT Clone 31282 Increases Intracellular Cytokine Production in TCells from Cancer Patient PBMC and Dissociated Tumour Cells (DTC)

In this example, intracellular flow cytometry staining was performed toassess the T cell cytokine production from freshly isolated matched PBMCand tumour infiltrated lymphocytes within dissociated tumour cells (DTC)from kidney carcinoma cancer patients. For DTC, tumours were mincedmechanically then incubated with Tumor Dissociation Kit (MiltenyiBiotech #130-095-929) under rotation in a gentleMACS dissociator,following manufacturer instructions for specific tumor types. Cells werestimulated for 16 h with a T cell stimulation bead cocktail (Dynabeads,Thermo Fisher) before performing intracellular staining. During the last3 hours of stimulation, protein Transport Inhibitor Cocktail(eBioscience) and Cell stimulation cocktail (eBioscience) were added tothe cells. Conjugated antibodies were purchased from Ebioscience/ThermoFisher Scientific, BioLegend or BD Biosciences. Surface staining wasperform per manufacturer's instruction using filtered FACS buffer (PBS+2mM EDTA+0.1% BSA) and Brilliant Stain buffer (BD #563794). Cells wereblocked with appropriate Human FcBlock (BD #564220) prior to surfacestaining. For intracellular staining, cells were fixed and permeabilizedusing BD Cytofix/cytoperm solution (BD Biosciences). Cells were stainedwith the following antibody panel: anti-CD45-BB515 (Clone HI30, BDHorizon 564585), anti-CD73-BV421 (Clone AD2, BD Horizon 562430),anti-CD8a-BV510 (Clone SK1, BD Horizon 563919), anti-CD3-BV650 (CloneSK7, BD Horizon 563999), anti-IFNγ-BV711 (Clone 4S.B3, BD Horizon564793), anti-IL-2-APC (MQ1-17H12, eBioscience 17-7029-82),anti-CD4-APC-R700 (Clone RPA-T4, BD Horizon 564975), LVD efluor 780(eBioscience 65-0865-14), anti-TIGIT-PE (Clone MBSA43, eBioscienceE13456-108), anti-CD39-PE-Dazzle594 (Clone A1, Biolegend 328224) andTNFα-PE-cy7 (Clone Mab11, eBioscience 25-7349-82). Acquisition wasperformed on a FACS Fortessa (BD Biosciences) and analyzed with FlowJosoftware (FlowJo, LLC). Viable cells were gated on Forward and Sidescatter. T cells subsets were gated as followed: CD45+CD3+ for PBMC andCD45+CD3+CD4+ and CD45+CD3+CD8+ for DTC. Cytokine-secreting T cells weregated using unstained and unstimulated controls.

FIG. 24D shows that intracellular content of IL2, IFNg and TNFa were allincreased upon activation in presence of a-TIGIT clone 31282. Thisincrease was observed in CD3+ T cells from PBMC in accordance with dataillustrated in FIG. 24C but also in both CD4+ and CD8+ TIL fromdissociated tumour cells. This demonstrates the potential of a-TIGITclone 31282 to increase the activation of PBMC and TIL populations fromcancer patient T cells.

Example 21: A-TIGIT Clone 31282 Induces Preferential Cytotoxicity ofTreg in PBMC from Cancer Patients

In this example, isolated PBMCs from a lung cancer patient wereresuspended in complete RPMI medium (supplemented with 10% FBS heatinactivated+50 U Penicillin+50 U Streptomycin). 2.5×105 human PBMCs weredistributed per well in 96 U well plate. Anti-human TIGIT antibody clone31282, human IgG1 isotype control (BioXcell BE0297) or Rituximab(InvivoGen hcd20-mab1) were added at a final concentration of 6.6 nM toeach corresponding well. Cells were incubated for 20 h at 37° C. with 5%CO2. Then cells were collected and stained with the following antibodypanel: LVD efluor 520 (eBioscience 65-0867-14), ant-TCRab-PercP-Cy5.5(Clone IP26, Biolegend 306723), anti-CD4-BV510 (Clone SK3, BD Horizon562970), anti-CD8-APC-Cy7 (Clone SK1, Biolegend 344714), anti-CD25-BV605(Clone 2A3, Biolegend 562660), anti-CD127-APC (A019D5, Biolegend351316), anti-CCR7-BV421 (Clone G043H7, Biolegend 353207) andanti-CD45RO-PE-Cy7 (Clone UCHL1, Biolegend 304229). Results arepresented on gated live cells. Absolute quantification is done usingAccuCheck Counting beads (Life technologies) following manufacturer'sspecifications. After calculation of absolute cell numbers per μl, % ofspecific lysis is calculated using the following formula=(1−(absolutenumber of cells per μl on 31282 TIGIT antibody treated sample/average oftriplicate of control isotype treated samples))×100. Results arepresented as mean % of specific lysis on triplicates+/−SD. The cytotoxicactivity of ADCC/ADCP effector cells was assessed by measuring % ofspecific cell lysis on gated CD19+ cells upon incubation with Rituximab.

As shown in FIG. 25 , anti-TIGIT clone 31282 triggers higher specificlysis on Tregs cells (30.1+/−3%) than on CD45RO+CCR7+/−CD8+ T cells(total memory CD8+ T cells) (−1.48+/−6%) or CD45RO+CCR7+/−CD4+ T (totalmemory CD4+ T cells) (0.64+/−3%). Rituximab positive control triggers77.9% (+/−6.8%) of specific lysis on gated CD19+ cells. Overall datademonstrate a preferential depletion of Treg cells from cancer patientPBMC as compared to total memory CD4+ and CD8+ T cell populations.Similar preferential depletion of Treg cells was observed using cellsfrom a patient with colon adenocarcinoma.

Example 22: Characterization of TIGIT Expression on Immune Populationsfrom Cancer Patient PBMC and Dissociated Tumour Cells

Flow cytometry analyses were performed to assess the expression of TIGITon immune cell subsets from freshly isolated matched PBMC and tumourinfiltrated lymphocytes within dissociated tumour cells (DTC) fromcancer patients. Samples from different indications were acquired:Ovarian cancer, Kidney cancer, HNSSC, Cutaneous carcinoma, Melanoma andLung cancer. For DTC, tumours were minced mechanically then incubatedwith Tumor Dissociation Kit (Miltenyi Biotech #130-095-929) underrotation in a gentleMACS dissociator, following manufacturerinstructions for specific tumour types. PBMC were isolated from wholeblood on a density gradient medium (Lymphoprep Axis-Shield #1115758).Phenotyping data were compared with frozen PBMC isolated from healthyindividuals (n=10).

Cells were stained per manufacturer's instruction using filtered FACSbuffer (PBS+2 mM EDTA+0.1% BSA) and Brilliant Stain buffer (BD #563794).Cells were blocked with appropriate Human FcBlock (BD #564220) prior tostaining and were fixed using IC fixation buffer (eBioscience#00-8222-49) prior acquisition. DTC were stained with the followingantibody panel: anti-CD45-BB515 (Clone HI30, BD Horizon 564585),anti-CD73-BV421 (Clone AD2, BD Horizon 562430), anti-CD8a-BV510 (CloneSK1, BD Horizon 563919), anti-CD3-BV650 (Clone SK7, BD Horizon 563999),anti-CD56-BV711 (Clone 5.1H11, Biolegend 362542), anti-CD279-BV785(Clone EH12.2H7, Biolegend 329930), anti-CD127-APC (Clone A019D5,Biolegend 351316), anti-CD4-APC-R700 (Clone RPA-T4, BD Horizon 564975),LVD efluor 780 (eBioscience 65-0865-14), anti-TIGIT-PE (Clone MBSA43,eBioscience E13456-108), anti-CD39-PE-Dazzle594 (Clone A1, Biolegend328224) and CD25-PE-cy7 (Clone BC96, Biolegend 302612). PBMC werestained with the following antibody panel: anti-CD45RO-BB515 (CloneUCHL1, BD Horizon 564529), anti-CD73-BV421 (Clone AD2, BD Horizon562430), anti-CD8a-BV510 (Clone SK1, BD Horizon 563919), anti-CD3-BV650(Clone SK7, BD Horizon 563999), anti-CD56-BV711 (Clone 5.1H11, Biolegend362542), anti-CD197-BV786 (Clone 3D12, BD Horizon 563710),anti-CD127-APC (Clone A019D5, Biolegend 351316), anti-CD4-APC-R700(Clone RPA-T4, BD Horizon 564975), LVD efluor 780 (eBioscience65-0865-14), anti-TIGIT-PE (Clone MBSA43, eBioscience E13456-108),anti-CD39-PE-Dazzle594 (Clone A1, Biolegend 328224) and CD25-PE-cy7 (Clbone BC96, Biolegend 302612). Acquisition was performed on a FACSFortessa (BD Biosciences) and analyzed with FlowJo software (FlowJo,LLC). Viable cells were gated on Forward and Side scatter. VariousImmune cells subsets were gated as followed: CD3+ CD4+ CD127+ CD25−(CD3+ CD4+ non-Treg cells), CD3+ CD4+ CD127low CD25+ (regulatory Tcells), CD3+ CD8+ (CD3+ CD8+ T cells), CD3− CD56+ (NK cells), CD3+ CD56+(NKT cells), CD3− CD56− (non-T/NK cells). Quantibrite PE beads (BD#340495) were run at the same instrument settings and used to convertfluorescence data into number of antibodies bound per cell.

Frequency of TIGIT expression on different immune populations isrepresented in FIG. 26A and TIGIT density for each subset is representedin FIG. 26B using Box and whiskers representation using the Tukey methodto compute percentiles.

Data show that TIGIT frequency on T cell subset is higher on PBMC fromcancer patients as compared to PBMC from healthy donors. This frequencyis further increased on DTC TILS (FIG. 26A). While the same observationis made looking at the density of TIGIT on the surface of CD3+ CD4+non-Treg cells and CD4+ Treg cells, for CD3+ CD8+ T cells the number ofTIGIT molecules per cell is decreased on DTC TILS (FIG. 26B).

Example 23: Structural and Functional Epitope Mapping of TIGIT and Clone31282

To further characterize and understand the interaction betweenanti-TIGIT mAb clone 31282 and TIGIT recombinant protein, the crystalstructure of 31282 in complex with TIGIT was determined by X-raydiffraction.

A. TIGIT and Fab Expression, Purification and Crystallization

Human TIGIT residues 23-128 was produced by Proteros Biostructures GmbH.TIGIT (23-128) with N-terminal HIS-tag (thrombin cleavable) was clonedinto pET15b and expressed in LB medium in B121(DE3) at 37° C. ininclusion bodies. Inclusion bodies (IBs) were washed with buffercontaining Tris/HCl pH 7.4 and Tris/HCl pH 7.4, 0.05% Brij-35. IBs weredenaturated with 6 M Gdm/HCl, 50 mM Tris pH 8.5 and 10 mM DTT. Refoldingwas performed in 50 mM Tris/HCl pH8, 1 mM GSH, 0.5 mM GSSG, 150 mM NaCl.Refolded protein was purified on HIS-trap. The N-terminal HIS-tag wasremoved via Thrombin cleavage and further purification on Superdex-75equilibrated in 50 mM Tris/HCl pH 7.5, 200 mM NaCl.

For Fab fragment expression, HEK293F cell were grown in Freestyle F17with 1% penicillin/streptomycin, 2 mM L-glutamine and 0.1% Pluronic.Expanded cultures for transfection were cultivated in 3 L Erlenmeyerflasks (Corning, 2 L cell culture working volume, 37° C., 8% v/v CO2,

80-120 rpm, 50 mm amplitude). The culture was diluted one day beforetransfection and the cell number adjusted to 1×106 cells/ml. The volumeof the expression culture was 6 L. A transient transfection wasperformed with plasmids for light and heavy chain of Fab. A MasterMix ofDNA/FectoPro (FectoPro, PolyPlus) was prepared in pure F17 Medium andincubated for 10 minutes (according to PolyPlus protocol). Thistransfection mix was added to the cell suspension dropwise and theBooster was added immediately. 18 hrs after transfection the culture wasfed with 3 g/L glucose.

For purification of the Fab fragment, 6 L supernatant of HEK293 cellculture was harvested by centrifugation 6 days after transfection andapplied to a 30 ml KappaSelect column. KappaSelect was washed with PBSpH 7.4, eluted with sodium citrate pH 3 and Fab containing fractionswere neutralized with Tris buffer. Fab was further purified on SuperdexS-200 column equilibrated in 20 mM Tris pH 8, 100 mM NaCl and stored at−80° C. until further use.

For the Fab-TIGIT complex formation, purified TIGIT was mixed withpurified Fab in a ratio of 1.5:1 and the complex was purified onSuperdex-200 equilibrated in 20 mM Tris pH 8, 100 mM NaCl. The Fab-TIGITcomplex was concentrated to 35 mg/ml for crystallization. The Fab-TIGITcomplex was crystallized at 277K using the vapour diffusion method bymixing 0.1 μl protein solution (35.3 mg/ml in 20 mM TRIS pH 8.0; 100 mMNaCl) in a 1:1 ratio with reservoir solution (0.10 M Sodium cacodylatepH 6.00; 15% (w/v) PEG4000). Crystals were cryo-protected by immersingthem in reservoir solution with 25% glycerol added.

B. Data Collection and Processing

A cryo-protocol was established using Proteros Biostructures GmbHStandard Protocols. Crystals have been flash-frozen and measured at atemperature of 100 K. X-ray diffraction data was collected fromFab:TIGIT complex crystals at the SWISS LIGHT SOURCE (SLS, Villigen,Switzerland) using cryogenic conditions. The crystals belong to spacegroup P1. Data were processed using the programmes XDS and XSCALE. Datacollection and processing statistics can be found in Table 22.

TABLE 22 Data collection and processing statistics X-Ray sourcePXII/X10SA (SLS¹) Wavelenght [Å] 1.0000 Detector PILATUS 6M Temperature[K] 100     Space group P1 Cell: a; b; c; [Å] 41.73; 71.46; 110.26 α; β;γ; [°] 96.7; 95.8; 106.5 Resolution [Å]   2.31 (2.56-2.31) Uniquereflections 50537 (13271) Multiplicity 2.0 (1.9) Completeness [%] 96.1(95.3) R_(sym) [%]  8.1 (43.5) R_(meas) [%] 11.0 (59.1) Mean(I)/sd³ 8.11(1.94) ¹SWISS LIGHT SOURCE (SLS, Villigen, Switzerland) ²values inparenthesis refer to the highest resolution bin ³calculated fromindependent reflections

C. Structure Modelling and Refinement

The phase information necessary to determine and analyse the structurewas obtained by molecular replacement. A previously solved structure ofFab was used as a search model. Subsequent model building and refinementwas performed according to standard protocols with the software packagesCCP4 and COOT. For the calculation of the free R-factor, a measure tocross-validate the correctness of the final model, about 2.5% ofmeasured reflections were excluded from the refinement procedure (seeTable 23).

TLS refinement (using REFMAC5, CCP4) has been carried out, whichresulted in lower R-factors and higher quality of the electron densitymap. Automatically generated local NCS restraints have been applied(keyword “ncsr local” of newer REFMAC5 versions). The ligandparameterisation and generation of the corresponding library files werecarried out with CHEMSKETCH and LIBCHECK (CCP4), respectively.

The water model was built with the “Find waters” algorithm of COOT byputting water molecules in peaks of the Fo-Fc map contoured at 3.0followed by refinement with REFMAC5 and checking all waters with thevalidation tool of COOT. The criteria for the list of suspected waterswere: B-factor greater 80 Å2, 2Fo-Fc map less than 1.2σ, distance toclosest contact less than 2.3 Å or more than 3.5 Å. The suspected watermolecules and those in the ligand binding site (distance to ligand lessthan 10 Å) were checked manually. The final complex structure wasrefined with PHENIX. We chose the refinement parameter including XYZcoordinates, Real space, Individual B-factors and Group B-factors.Optimize X-ray/stereochemistry weight and NCS restraints were alsochosen for refinement. The Ramachandran Plot of the final model shows95.39% of all residues in the preferred region, 3.95% in the allowedregion. Statistics of the final structure and the refinement process arelisted in Table 23.

TABLE 23 Refinement statistics¹ Resolution [Å] 108.40-2.31 Number ofreflections (working/test) 49289/1247 R_(work) 0.2025 R_(free) [%]0.2466 Total number of atoms: Protein 8282 Water 676 Deviation fromideal geometry: ³ Bond lengths [Å] 0.003 Bond angles [deg] 0.771Ramachandran plot: ² Preferred regions [%] 95.39 Allowed regions [%]3.95 Disallowed regions [%] 0.66 ¹values as defined in PHENIX ²Calculated with COOT

D. Overall Structure

The heavy and light chains of the human Fab antibody fragment show thetypical folding of human antibodies (FIG. 27A). There are twohetero-trimers in the asymmetric unit with basically the same overallconformation. The model comprises residues 23 to 128 of TIGIT, residues1 to 224 of the heavy chain of clone 31282 and residues 1 to 214 of thelight chain of clone 31282. One short loop region of the heavy chain isnot fully defined by electron density and has thus not been included inthe model.

Diffraction images were analysed using FoldX program to estimate energycontribution of residues and define interaction hotspots. The amino acidresidues forming the binding interface are well defined in the electrondensity map. The interpreted X-ray diffraction data show clearly theinteractions between the Fab and TIGIT (FIGS. 27B and 27C). Clone 31282light chain CDR are interacting with 2 regions of TIGIT with CDR L1Arg30 and Tyr33 contacting TIGIT residues Asn58 and Glu60; with CDR L1Arg30 and CDR L3 Phe93 contacting TIGIT residue Ile109. CDR L2 has nocontact with TIGIT (Table 24). Clone 31282 heavy chain interacts withdifferent regions of TIGIT with CDR H1 Tyr33 contacting TIGIT on residueLeu73; with CDR H2 Val50, Ser54 and Ser57 contacting TIGIT on residueLeu73; with CDR H3 Asp102, Tyr103 and Trp104 contacting TIGIT on residueGln56, Ile68, Leu73 and His76.

Based on this crystal structure of the a-TIGIT clone 31282/TIGITcomplex, the residues of TIGIT that are contacted by clone 31282(epitope residues for TIGIT bound by clone 31282) and the residues ofclone 31282 that are contacted by TIGIT (paratope residues for clone31282 bound by TIGIT) were determined. Tables 24 and 25 and FIG. 27Cshow the residues of TIGIT in contact with the light (Table 24) or heavy(Table 25) chain residues of clone 31282. Contact residues were definedas each amino acid meeting each of the following criteria: (i) it has acalculated binding free energy contribution greater than 0.3 kcal/mol,(ii) it has an experimental averaged B-factor lower than the meanB-factor of all residues in the X-ray structure, (iii) it makes at least3 pairs of heavy-atom interatomic contacts with antibody atoms at adistance less than or equal to 4.0 Angstroms, (iv) it does not make onlysolvent-exposed hydrogen bond or ionic interactions, (v) if it is anon-aromatic polar residue (Asn, Gln, Ser, Thr, Asp, Glu, Lys, or Arg),it makes at least one hydrogen bond or ionic interaction with theantibody.

TABLE 24 Summary of epitope residues of TIGIT and corresponding paratoperesidues on the light chain of clone 31282 Clone 31282 Amino Acid TIGITAmino Acid Light Chain Asn 58 Tyr 33 Glu 60 Arg 30 Tyr 33 Ile 109 Arg30Phe 93

TABLE 25 Summary of epitope residues of TIGIT and corresponding paratoperesidues on the heavy chain of clone 31282 Clone 31282 Amino Acid TIGITAmino Acid Heavy Chain Gln 56 Trp 104 Ile 68 Tyr 103 Trp104 Leu 73 Tyr33Val50 Ser 54 Val 50 His 76 Asp 102 Tyr 103 Trp104

Example 24: Competition Assay Between a-TIGIT Clones 31282 and 32959

Anti-TIGIT antibody clone 32959 of human IgG1 isotype was produced inHEK cells and purified as described in Example 17 above.

Jurkat cells overexpressing human TIGIT (Jurkat-hTIGIT) were collectedand distributed at 5.104 cells/well and incubated with antagonista-TIGIT clone 31282 at the following concentrations: 0 nM (No Ab), 0.08nM, 0.16 nM, 0.8 nM and 8 nM that represent a range of concentrationfrom 0 to 100 times the Kd of this clone. Excess of antibody was washed,and cells were incubated with decreasing concentration (8; 4; 2; 1; 0.5;0.25; 0.125; 0.062; 0.031; 0.016; 0.008 and 0.004 nM) of directlycoupled (AF647) anti-TIGIT clone 32959 for 30 min at 4° C. Geometricmean fluorescence intensity was analysed using LSR BD Fortessa. Cellbinding was recorded as the median florescence intensity of AF647. Forcalculation of EC50 binding of clone 32959, the half-maximalconcentration of binding (EC50) to hTIGIT-Jurkat was calculated using afour-variable curve-fit equation in Prism, and the obtained values areshown in Table 26 and illustrated in FIG. 28 . The results show a strongbinding of a-TIGIT clone 32959, independently of the concentration ofclone 31282, demonstrating the absence of competition with an antagonista-TIGIT antibody.

TABLE 26 EC₅₀ concentration for binding of a-TIGIT clone 32959 toJurkat-hTIGIT in presence of increasing concentration of antagonista-TIGIT clone 31282 a-TIGIT a-TIGIT a-TIGIT a-TIGIT a-TIGIT 31282 3128231282 31282 31282 at at at at at 0 nM 0.08 nM 0.16 nM 0.8 nM 8 nM EC₅₀(nM) 0.22 0.33 0.37 0.49 0.39 binding for a-TIGIT clone 32959 Cellbinding 588 Jurkat Human TIGIT FON (Fold Over Negative) for a-TIGITclone 32959

Example 25: Determination of Pharmacokinetic Profile of Clone 31282after Single i.v. Injection in Cynomolgus Monkey

Cynomolgus monkeys received a-TIGIT clone 31282 IgG1 or IgG4 via i.v.bolus injection. Antibody was administered at 3 different concentrations(0.1 mg/kg; 1 mg/kg; 10 mg/kg) to 2 animals (1 male and 1 female). Bloodwas collected through 504 hours post-dose on Day 1. Blood samples wereprocessed for plasma and analyzed for concentration of a-TIGIT clone31282 IgG1 or IgG4 using an ELISA method. Plasma concentration-time datafrom individual animals were used to calculate toxicokinetic parametervalues for a-TIGIT clone 31282 IgG1 and IgG4 after IV dosing using theintravascular model in Phoenix WinNonlin (version 6.3, Pharsight, aCertara Company, Princeton, N.J.).

Following IV bolus dosing of a-TIGIT clone 31282 IgG1 and IgG4 at 0.1,1, and 10 mg/kg, IgG1 concentrations were quantifiable in plasma of maleand female monkeys through 240 h, 336 h, and 504 h post-dose,respectively, and IgG4 was quantifiable through 168 h, 240 h, and 504 h,respectively (FIG. 29 and Table 27). There were no apparent sex-relateddifferences in systemic exposure (Cmax and AUClast) to IgG1 and IgG4after i.v. bolus dosing of a-TIGIT clone 31282 IgG1 or IgG4, with ratios(males/females) ranging from 0.855 to 1.16.

Following i.v. bolus dosing of a-TIGIT clone 31282 IgG1 to male andfemale monkeys, plasma IgG1 concentrations declined biphasically at alldose levels, with mean terminal half-life (t½) ranging from 84.7-174 h(FIG. 29 ). Systemic clearance (CL) was consistent across the dosesstudied, ranging from 0.280 to 0.392 mL/h/kg. Apparent volume ofdistribution at steady state (Vss) was consistent among the dose levelstested, with values ranging from 53.7-66.5 mL/kg. The 10-fold increasesin a-TIGIT clone 31282 IgG1 dose in the range of 0.1 to 1 mg/kg and from1 to 10 mg/kg resulted in approximately proportional increases inexposure (9.57- to 14.5-fold increases).

Following i.v. administration of a-TIGIT clone 31282 IgG4 to male andfemale monkeys, plasma IgG4 concentrations declined biphasically at alldose levels tested, with t½ of 148-334 h (FIG. 29 and Table 28). CL wasconsistent among the dose levels tested, ranging from 0.160 to 0.219mL/h/kg. Mean Vss ranged from 41.2-70.7 mL/kg. The 10-fold increases ina-TIGIT clone 31282 IgG4 dose in the range of 0.1 to 1 mg/kg and from 1to 10 mg/kg resulted in approximately proportional increases in exposureto IgG4 (9.32- to 12.5-fold increases).

TABLE 27 Summary of mean Toxicokinetics parameters for a-TIGIT clone31282 human IgG1 after i.v. bolus in Cynomolgus monkey a-TIGIT clone31282 human IgG1 Dose (mg/kg) 0.1 1 10 C_(max) (ug/ml) 2.34 22.4 268t_(max) (h) 1 1 1 AUC_(last) (h*ug/ml) 224 2330 33700 t_(1/2) (h) 17484.7 111 Cl (mL/h/kg) 0.292 0.392 0.280 V_(ss) (mL/kg) 66.5 57.2 53.7

TABLE 28 Summary of mean Toxicokinetics parameters for a-TIGIT clone31282 human IgG4 after i.v. bolus in Cynomolgus monkey a-TIGIT clone31282 human IgG4 Dose (mg/kg) 0.1 1 10 C_(max) (ug/ml) 2.81 26.2 283t_(max) (h) 1 1 1 AUC_(last) (h*ug/ml) 238 2690 39100 t_(1/2) (h) 251334 182 Cl (mL/h/kg) 0.190 0.160 0.216 V_(ss) (mL/kg) 65.7 70.7 57.5

Example 26: Characterization of TIGIT Expression on Human Tumour CellPopulations

Flow cytometry analyses were performed to assess the expression of TIGITon normal and tumour T or B cells in blood samples from cancer patientswith different indication of blood cancer.

Sézary Syndrome patient samples were tested to compare TIGIT expressionon malignant and normal CD4+ T cell populations. To separate thesepopulations, a pre-determination of the malignant clone TCR-Vbrearrangement was performed using Beckman Coulter TCR-Vb repertoire kit(#IM3497). Once the malignant clone was identified, TIGIT expression wasprofiled on immune cells of Sézary Syndrome patients using the followingcommercial reagents: anti-CD3 Krome Orange (#B00068), anti-CD4-PE(#A07751), anti, CD8-PC7 (#737661), anti-CD56-PC5 (#A07789),anti-CD45-Pacific Blue (#A74763), anti-CD19-AF750 (#A94681) andanti-Vb8-FITC (#IM1233) (all from Beckman-Coulter) and anti-TIGIT-APC(clone MBSA43, ebiosciences #17-9500-42). Flow-cytometry analyses ofSézary Syndrome patient samples were performed on a CytoFlex apparatus(Beckman-Coulter). Data were analyzed with FloJo software (FlowJo, LLC).

A representative example is shown on FIG. 30A. Gating strategy for thisdonor that has a malignant TCR-Vb8 clone is shown in FIG. 30A withmalignant cells being CD45+CD3+CD4+Vb8+ and normal CD4+ T cells beingCD45+CD3+CD4+Vb8−. A strong expression of TIGIT is observed on themalignant CD4+ T cells compared to the normal CD4+ T cells (respectiveMFI of 4987 and 999) (FIG. 30B).

Similarly, flow cytometry analyses were performed to assess theexpression of TIGIT on normal and malignant B cells in bone marrowsamples from patients with CLL. The samples were stained with thefollowing antibody panel: LVD efluor 780 (eBioscience 65-0865-14),anti-CD45-BB515 (Clone HI30, BD Horizon 564585), anti-CD5-BV510 (CloneUCHT2, Biolegend 363381), anti-CD19-BV711 (Clone SJ25C1, BD Horizon563036) and anti-TIGIT-PE (Clone MBSA43, eBioscience E13456-108).Acquisition was performed on FACS Fortessa (BD Biosciences) and analyzedwith FlowJo software (FlowJo, LLC). Viable cells were gated on Forwardand Side scatter. Various cell-subsets were gated as followed:CD45+CD19+CD5-(Normal B cells) and CD45+CD19+CD5+(Malignant B-CLL).

A representative example is shown on FIG. 31 with gating strategyillustrated in FIG. 31A. A high proportion of malignant B-CLL cells arepositive for TIGIT (75%), in contrast to normal B cells (1%) (MFIs of1440 and 810, respectively) (FIG. 31B).

Overall, the data obtained demonstrate that tumour cells express TIGITin specific blood cancer indications.

Example 27: Anti-Tumour Activity of Anti-TIGIT Antagonistic Antibody inMonotherapy in Mouse T Cell Lymphoma Model

For this experiment, EL4 T cell lymphoma cells (ATCC® TIB-39™) wereengineered to stably express mouse TIGIT (EL4-mTIGIT). EL4 cellstransduced with a similar vector coding for GFP were used as control(EL4-GFP). Pools of cells were subcloned to obtain clones of EL4-mTIGITand EL4-GFP. The anti-TIGIT antibody used was a modified version ofantibody 29527 (modified such that residue 27 of VH FR3 is mutated fromL to V and where residue 6 of VH FR4 is mutated from M to T) andproduced on a human IgG1 isotype. Female Balb/c mice of 8 weeks wereinoculated with 1,000,000 EL4-mTIGIT cells or 200,000 EL4-GFP cellssubcutaneously. On day 7 after inoculation, when tumor volumes were onaverage around 110 mm3, mice were randomized in treatment groups withequal tumor volume (n=15 per group for EL4-mTIGIT and n=10 per group forEL4-GFP). Mice were treated with 200 μg of anti-TIGIT or isotype controlantibody (hIgG1, BioXcell) by intraperitoneal injections on day 7, day10, day 13 and day 16 after tumour inoculation. Tumor growth wasmonitored and tumor volumes were measured with electronic calipers threetimes a week from day 7 until day 26. Mice were sacrificed when tumorvolume exceeded 2000 mm3. Tumor growth curves were statisticallyanalyzed by a linear mixed model. Differences between treatment groupswere evaluated by testing the interaction of time*treatment group.

FIG. 32 illustrates tumor growth curves in mice inoculated withEL4-mTIGIT (A-C) or EL4-GFP (D-F). Median tumor growth curves (A and D)as well as individual tumor growth curves for mice treated with hIgG1isotype control (B and E) or antagonist a-TIGIT Ab (C and F) arerepresented. In mice inoculated with EL4-mTIGIT cells, there was asignificant suppression of tumor growth when treated with anti-TIGIT Abcompared to isotype control treated group (p<0.001). Whereas in thegroup treated with isotype control antibody, 3 out of 15 micedemonstrated a control of tumor growth with a volume below 700 mm3 atthe end of the model, this number was increased to 8 out of 15 mice inthe group treated with antagonist anti-TIGIT antibody. No anti-tumorefficacy or complete response could be observed in EL4-GFP tumor bearingmice when comparing antagonist a-TIGIT treatment to isotype controlantibody. Together, these data demonstrate that antagonist a-TIGITantibody (hIgG1) has significant antitumor efficacy in a model withtumor cells expressing TIGIT.

Example 28: Anti-Tumour Activity of Anti-TIGIT Antagonistic Antibody inCombination with Immune Checkpoint Antibodies in CT26 Colon CarcinomaMouse Models

In addition to the combination of anti-TIGIT Ab with an anti-PD1antibody (Examples 12, 13 and 14), the antitumor efficacy of ananti-TIGIT antibody was also evaluated in combination with agonistantibodies specific for co-stimulatory molecules 4-1BB, OX40 and GITR,as well as with an antagonist antibody specific for checkpointinhibitory molecule ICOS.

CT26 tumour cell line was purchased from ATCC® (CRL-2638™). Femalebalb/c mice of 8 weeks were subcutaneously inoculated in the right flankwith 500.000 cells. On day 9 after inoculation, when tumor volumes wereon average around 75 mm3, mice were randomized in treatment groups withequal tumor volume (n=10 mice per group). All the antibodies were givenintraperitoneally every 3 days starting on the day of randomization fora total of 3 injections. The anti-TIGIT antibody used was a modifiedversion of antibody 29527 (modified such that residue 27 of VH FR3 ismutated from L to V and where residue 6 of VH FR4 is mutated from M toT) produced on a mouse IgG2a isotype, that was given at 20 μg/mouse.Anti-4-1BB (clone 3H3, BioXCell, BE0239) was given at 5 ug/mouse,a-OX-40 (clone OX-86, BioXCell, BE0031) was given at 20 ug/mouse, a-GITR(clones DTA-1, BioXCell, BE0063) was given at 10 ug/mouse; and a-ICOS(clone 7E.17G9, BioXCell, BE0059) was given at 200 ug/mouse. Tumorgrowth was monitored and tumor volumes were measured with electroniccalipers three times a week from day 7 until day 35. Mice weresacrificed when tumor volume exceeded 2000 mm3. Tumor growth curves werestatistically analyzed by a linear mixed model on logarithmicallytransformed tumor volumes. Differences between treatment groups wereevaluated by testing the interaction of time*treatment group. Thisresulted in a good model fit for the vast majority of the data, exceptfor very small tumor volumes (below 10 mm3). Therefore, these smalltumor volumes were treated as missing values. To test for a synergisticeffect arising from combining the anti-TIGIT antibody with thecorresponding immune checkpoint antibody (IC—i.e. anti-41BB, anti-OX40,anti-GITR, and anti-ICOS), treatment groups were re-coded by acombination of two variables; anti-TIGIT (yes/no) and IC (yes/no). Asynergistic effect, on top of the additive effect of each treatment(anti-TIGIT*time and IC*time) was evaluated by testing the interactionterm anti-TIGIT*IC*time.

FIG. 33A shows median tumor growth curves per group as well asindividual growth curves for mice treated by anti-TIGIT in monotherapyor in combination with anti-4-1BB. There was significant suppression oftumor growth in mice treated with anti-TIGIT+anti-4-1BB compared toanti-TIGIT or anti-4-1BB monotherapy (p=0.0005 and p<0.0001respectively). The combination of anti-TIGIT and anti-4-1BB antibodiesresulted in 6/10 mice showing a complete response (where tumor is <30mm3 and considered as non-measurable), as compared with 1/10 or 0/10complete response in groups treated respectively with a-TIGIT or a-4-1BBas a single agent. These data demonstrate the significant anti-tumorefficacy of anti-TIGIT therapy in combination with anti-4-1BB fortreatment of pre-established tumors.

FIG. 33B shows median tumor growth curves per group as well asindividual growth curves for mice treated by anti-TIGIT in monotherapyor in combination with anti-OX-40. There was significant suppression oftumor growth in mice treated with anti-TIGIT+anti-OX-40 compared toanti-TIGIT or anti-OX-40 monotherapy (p=0.0002 and p<0.0001,respectively). The combination of anti-TIGIT+anti-OX-40 achievedsynergistic anti-tumor efficacy that was more than the additive effectof both monotherapy treatments (p=0.02). The combination of anti-TIGITand anti-OX-40 antibodies resulted in 7/10 mice showing a completeresponse as compared with 1/10 or 0/10 complete response in groupstreated respectively with a-TIGIT or a-OX-40 as a single agent. Thesedata demonstrate the significant and synergistic anti-tumor efficacy ofanti-TIGIT therapy in combination with anti-OX-40 for treatment ofpre-established tumors.

FIG. 33C shows median tumor growth curves per group as well asindividual growth curves for mice treated by anti-TIGIT in monotherapyor in combination with anti-GITR. There was significant suppression oftumor growth in mice treated with anti-TIGIT+anti-GITR compared toanti-TIGIT or anti-GITR monotherapy (p<0.0001). The combination ofanti-TIGIT+anti-GITR achieved synergistic anti-tumor efficacy that wasmore than the additive effect of both monotherapy treatments (p=0.01).The combination of anti-TIGIT and anti-GITR antibodies resulted in 6/10mice showing a complete response as compared with 1/10 or 0/10 in groupstreated respectively with anti-TIGIT or anti-GITR as a single agent.These data demonstrate the significant and synergistic anti-tumorefficacy of anti-TIGIT therapy in combination with anti-GITR fortreatment of pre-established tumors.

FIG. 33D shows median tumor growth curves per group as well asindividual growth curves for mice treated by anti-TIGIT in monotherapyor in combination with anti-ICOS. There was significant suppression oftumor growth in mice treated with anti-TIGIT+anti-ICOS compared toanti-TIGIT or anti-ICOS monotherapy (p=0.003 and p=0.0001 respectively).The combination of anti-TIGIT and anti-ICOS antibodies resulted in 1/10mice showing a complete response (where tumor is <30 mm3 and consideredas non-measurable), as compared with 1/10 or 0/10 in groups treatedrespectively with anti-TIGIT or anti-ICOS antibodies as a single agent.These data demonstrate the significant and synergistic anti-tumorefficacy of anti-TIGIT therapy in combination with anti-ICOS fortreatment of pre-established tumors.

Example 29: Activity of Anti-TIGIT Antagonistic Antibody on 165 T Cells

γδ (gamma-delta, or g/d) T cells are a population of unconventional Tcells with described antitumor activity (Zhao et al. 2018. J Transl Med.16:122) and antiviral activity (e.g. CMV infection) and also have beenimplicated in autoimmune diseases (Malik S et al. 2016. Front Immunol.7:14).

Flow cytometry analyses were performed to assess the expression of TIGITon γδ T cells on PBMC freshly isolated from healthy individuals with aseronegative or seropositive status for Cytomegalovirus (CMV) (CMVstatus was assessed by the EFS Nouvelle Aquitaine, Bordeaux, France).Cells were stained per manufacturer's instruction using filtered FACSbuffer (PBS+2 mM EDTA+0.1% BSA). Acquisition was performed on a FACSFortessa (BD Biosciences) and analyzed with BD FACS DIVA software (BDBiosciences). Cells were gated on Forward and Side scatter andviability. γδ T cells were gated as follows: CD3+ TCRγδ+Vδ2-(Vδ2− γδ Tcells) using the following antibodies: anti-TCR γδ APC, clone REA591#130 280 from Miltenyi; anti-TCR Vδ2-PE-Vio 770, clone REA771,#130-111-012 from Miltenyi; BV421 mouse anti-human CD3, clone UCHT1,#560365 from BD Biosciences; Zombie Aqua Fixable viable kit, #423101from Biolegend.

Similar to conventional αβ T cells, non-conventional Vδ2− γδ T cellsexpress TIGIT in both CMV negative and positive human populations(anti-TIGIT, clone MBSA43, #12-98500-42 from eBioscience) (FIG. 34A). Tocharacterize the functional consequence of blocking TIGIT receptor onthis cell population, magnetically isolated Vδ1+ γδ T cells (anti-TCRVd1-FITC, clone REA173 #130-100-532 and anti-FITC Microbeads#130-048-701 both from Miltenyi) or total PBMC from CMV positive donorswere activated with anti-Vδ1 (10 ug/ml) (clone R9.1, #IM1761 fromBeckman Coulter) and IL-15 (20 ng/ml), #200-15-50UG from Peprotech),IL-2 (100 U/ml, #200-02-1MG Peprotech) was additionally added toisolated Vδ1+γδ T cells, in presence or absence of TIGIT-ligand CD155(#9174-CD-050 from R&D Systems). FIG. 34B shows a dose-dependentdecrease in IFNγ secretion (ELISA kit, #3420-1 h-20 from Mabtech)mediated by the addition of TIGIT-ligand CD155 (0, 0.1, 1 and 10 μg/ml)with a maximal inhibition reached at 1 ug/ml of CD155. The addition ofanti-TIGIT Ab clone 31282 (10 ug/ml) fully restores IFNγ production tolevel equal or higher to the condition without CD155 ligand while humanIgG1 isotype control has very limited effect. FIG. 34C demonstratessimilar inhibitory effect mediated by CD155 (10 μg/ml) after anti-Vδ1activation of total PBMC and a total restoration of IFNγ secretion whena-TIGIT clone 31282 is added to the mix. These data demonstrate that,similar to αβ T cells, activity of γδ T cells can be impaired byligation of CD155 to TIGIT and that anti-TIGIT antibodies fully preventthis inhibition.

1-52. (canceled)
 53. An isolated antibody or antigen binding fragmentthereof that specifically binds to human TIGIT, the antibody or antigenbinding fragment comprising: a) amino acid sequences of complementaritydetermining regions (CDRs), HCDR1, HCDR2, and HCDR3, of a heavy chainvariable domain (VH) comprising the amino acid sequence of SEQ ID NO:221; and b) amino acid sequences of CDRs, LCDR1 and LCDR3, of a lightchain variable domain (VL) comprising the amino acid sequence of SEQ IDNO:
 222. 54. The isolated antibody or antigen binding fragment accordingto claim 53, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3,LCDR1 and LCDR3 is defined according to the method of Kabat, Chothia, orMacCallum.
 55. The isolated antibody or antigen binding fragmentaccording to claim 53, wherein the HCDR1 comprises amino acid residuesselected from the group consisting of: amino acid residues 27-35, aminoacid residues 31-35, amino acid residues 26-32, and amino acid residues30-35 of SEQ ID NO:
 221. 56. The isolated antibody or antigen bindingfragment according to claim 53, wherein the HCDR2 comprises amino acidresidues selected from the group consisting of: amino acid residues51-58, amino acid residues 50-65, amino acid residues 53-55, and aminoacid residues 47-58 of SEQ ID NO:
 221. 57. The isolated antibody orantigen binding fragment according to claim 53, wherein the HCDR3comprises amino acid residues selected from the group consisting of:amino acid residues 97-110, amino acid residues 95-102, amino acidresidues 96-101, and amino acid residues 93-101 of SEQ ID NO:
 221. 58.The isolated antibody or antigen binding fragment according to claim 53,wherein the LCDR1 comprises amino acid residues selected from the groupconsisting of: amino acid residues 24-35, amino acid residues 24-34,amino acid residues 26-32, and amino acid residues 30-36 of SEQ ID NO:222.
 59. The isolated antibody or antigen binding fragment according toclaim 53, wherein the LCDR3 comprises amino acid residues selected fromthe group consisting of: amino acid residues 90-98, amino acid residues89-97, amino acid residues 91-96, and amino acid residues 89-96 of SEQID NO:
 222. 60. The isolated antibody or antigen binding fragmentaccording to claim 53, wherein the antibody or antigen binding fragmentfurther comprises an LCDR2 amino acid sequence of a VL comprising theamino acid sequence of SEQ ID NO:
 222. 61. The isolated antibody orantigen binding fragment according to claim 60, wherein the LCDR2comprises amino acid residues selected from the group consisting of:amino acid residues 51-57, amino acid residues 50-56, amino acidresidues 50-52, and amino acid residues 46-55 of SEQ ID NO:
 222. 62. Theisolated antibody or antigen binding fragment according to claim 53,wherein the antibody or antigen binding fragment further comprises amethionine (M) to threonine (T) substitution at amino acid residue 116in VH framework 4 (FR4) region, wherein the M-T substitution improvesstability of the antibody or antigen binding fragment compared to anantibody or antigen binding fragment not comprising the substitution.63. The isolated antibody or antigen binding fragment thereof accordingto claim 53, wherein the antibody or antigen binding fragment comprisesa fragment of a human immunoglobulin.
 64. The isolated antibody orantigen binding fragment thereof according to claim 63, wherein thefragment of a human immunoglobulin is a fragment of human IgG.
 65. Theisolated antibody or antigen binding fragment thereof according to claim53, wherein the antibody is a human IgG1 antibody.
 66. The isolatedantibody or antigen binding fragment according to claim 53, wherein theantibody or antigen binding fragment selectively depletesTIGIT-expressing Treg cells.
 67. The isolated antibody or antigenbinding fragment according to claim 53, wherein the antibody or antigenbinding fragment decreases expression of TIGIT on CD8 T cells and/or onTreg cells.
 68. A pharmaceutical composition comprising an effectiveamount of the isolated antibody or antigen binding fragment according toclaim 53, and a pharmaceutically acceptable carrier.
 69. Thepharmaceutical composition of claim 68, wherein the antibody or antigenbinding fragment comprises a fragment of a human immunoglobulin, whereinthe fragment of a human immunoglobulin is a fragment of human IgG. 70.The pharmaceutical composition of claim 68, wherein the antibody is ahuman IgG1 antibody.
 71. The pharmaceutical composition of claim 68,wherein the antibody or antigen binding fragment selectively depletesTIGIT-expressing Treg cells.
 72. The pharmaceutical composition of claim68, wherein the antibody or antigen binding fragment decreasesexpression of TIGIT on CD8 T cells and/or on Treg cells.